BioMicroCenter:RNA HTL - Revision history

Hilo1: /* 3'Digital Gene Expression */

2025-06-09T19:26:10Z

3'Digital Gene Expression

← Older revision		Revision as of 19:26, 9 June 2025
Line 283:		Line 283:

	:Analysis of HT3DGE is not a standard method supported by most open source platforms. We have built pipelines to work with this data, but working closely with the Bioinformatics team is highly recommended.		:Analysis of HT3DGE is not a standard method supported by most open source platforms. We have built pipelines to work with this data, but working closely with the Bioinformatics team is highly recommended.
	\|<br>[[image:DGE_workflow.jpg\|right\|Standard 3'DGE workflow.]]		\|<br>[[image:DGE_workflow.jpg\|right\|Standard 3'DGE workflow.]] <br><br>
	[[image:HT3DGE_gene_example.jpg\|right\|Example 3'DGE Data]]<BR><BR>		[[image:HT3DGE_gene_example.jpg\|right\|Example 3'DGE Data]]<BR><BR>
	\|		\|

Hilo1: /* 3'Digital Gene Expression */

2025-06-09T19:25:52Z

3'Digital Gene Expression

← Older revision		Revision as of 19:25, 9 June 2025
Line 283:		Line 283:

	:Analysis of HT3DGE is not a standard method supported by most open source platforms. We have built pipelines to work with this data, but working closely with the Bioinformatics team is highly recommended.		:Analysis of HT3DGE is not a standard method supported by most open source platforms. We have built pipelines to work with this data, but working closely with the Bioinformatics team is highly recommended.
	\|<br>[[image:DGE_workflow.jpg~~\|thumb~~\|right~~\|275px~~\|Standard 3'DGE workflow.]]		\|<br>[[image:DGE_workflow.jpg\|right\|Standard 3'DGE workflow.]]
	[[image:HT3DGE_gene_example.jpg~~\|thumb~~\|right~~\|400px~~\|Example 3'DGE Data]]<BR><BR>		[[image:HT3DGE_gene_example.jpg\|right\|Example 3'DGE Data]]<BR><BR>
	\|		\|
	\|}		\|}

Hilo1: /* High-throughput RNA Library Preparation */

2025-06-09T19:25:10Z

High-throughput RNA Library Preparation

← Older revision		Revision as of 19:25, 9 June 2025
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	{{BioMicroCenter}}		{{BioMicroCenter}}
	= High-throughput RNA Library Preparation =		= High-throughput RNA Library Preparation =
	The BioMicro Center offers high-throuhgput RNA library preparation [[BioMicroCenter:FAQ#STANDARD_versus_HIGH_THROUGHPUT_LIBRARY_PREPARATION\|(HTL-RNA)]] for both standard input and low input samples. Together, these high-throughput methodologies provide a comprehensive set of options to address a diverse range of experiments investigating RNA biology. High-throughput library generation focuses on reducing price and processing time on a per sample basis. To do this, there are several key differences from our standard library preparation service. First, HTL-RNA services have set batch sizes (24 and 96) with fixed price points (i.e. charge for 16 samples will be the as 24 samples; similarly for 80 samples as 96). If under these batch sizes, DO MORE REPLICATES to simultaneously enhance experimental power and reduce price per sample. Some samples may fail and the cost of re-prepping those samples is NOT included in the quoted cost. Re-prep is only deemed necessary by the core where we have found evidence of a reagent failure. User request re-preps of individuals samples can be done by hand, but at a higher rate. HTL focuses on reducing price and processing time per sample, so some routine services provided with standard library preparation - such as initial and final quality control for all samples, sample arraying, or automatic re-prep of failed samples - are not built in. These services are available as add ons to the original service request. <br><br>

	[[Image:DE_HTL_RNA.jpeg\|thumb\|right\|400px\|Increased biological replicates increases power of analysis. (Liu et al. 2014 Bioinformatics 30(3): 301-4)]]		[[Image:DE_HTL_RNA.jpeg\|thumb\|right\|400px\|Increased biological replicates increases the power of analysis. (Liu et al. 2014 Bioinformatics 30(3): 301-4)]]

			The BioMicro Center offers high-throughput RNA library preparation [[BioMicroCenter:FAQ#STANDARD_versus_HIGH_THROUGHPUT_LIBRARY_PREPARATION\|(HTL-RNA)]] for both standard input and low input samples. Together, these high-throughput methodologies provide a comprehensive set of options to address a diverse range of experiments investigating RNA biology. High-throughput library generation focuses on reducing price and processing time on a per sample basis. To do this, there are several key differences from our standard library preparation service. First, HTL-RNA services have set batch sizes (24 and 96) with fixed price points (i.e. charge for 16 samples will be the as 24 samples; similarly for 80 samples as 96). If under these batch sizes, DO MORE REPLICATES to simultaneously enhance experimental power and reduce price per sample. Some samples may fail and the cost of re-prepping those samples is NOT included in the quoted cost. Re-prep is only deemed necessary by the core where we have found evidence of a reagent failure. User request re-preps of individuals samples can be done by hand, but at a higher rate. HTL focuses on reducing price and processing time per sample, so some routine services provided with standard library preparation - such as initial and final quality control for all samples, sample arraying, or automatic re-prep of failed samples - are not built in. These services are available as add ons to the original service request. <br><br>

	<b>Reminder: </b> high throughput projects are treated with <b>one condition</b> (PCR cycles, fragmentation time, etc...). They are meant for allowing high replicates of the same samples. High throughput submissions for projects with multiple sub-projects from separate individuals combined on the same plate result in a low success rate due to the different conditions for each sub project. Therefore, htl submissions from multiple projects will tolerate and may expect, failure rates well over 10%.		<b>Reminder: </b> high throughput projects are treated with <b>one condition</b> (PCR cycles, fragmentation time, etc...). They are meant for allowing high replicates of the same samples. High throughput submissions for projects with multiple sub-projects from separate individuals combined on the same plate result in a low success rate due to the different conditions for each sub project. Therefore, htl submissions from multiple projects will tolerate and may expect, failure rates well over 10%.

Hilo1: /* High-throughput RNA Library Preparation */

2025-06-09T19:23:49Z

High-throughput RNA Library Preparation

← Older revision		Revision as of 19:23, 9 June 2025
Line 2:		Line 2:
	= High-throughput RNA Library Preparation =		= High-throughput RNA Library Preparation =
	The BioMicro Center offers high-throuhgput RNA library preparation [[BioMicroCenter:FAQ#STANDARD_versus_HIGH_THROUGHPUT_LIBRARY_PREPARATION\|(HTL-RNA)]] for both standard input and low input samples. Together, these high-throughput methodologies provide a comprehensive set of options to address a diverse range of experiments investigating RNA biology. High-throughput library generation focuses on reducing price and processing time on a per sample basis. To do this, there are several key differences from our standard library preparation service. First, HTL-RNA services have set batch sizes (24 and 96) with fixed price points (i.e. charge for 16 samples will be the as 24 samples; similarly for 80 samples as 96). If under these batch sizes, DO MORE REPLICATES to simultaneously enhance experimental power and reduce price per sample. Some samples may fail and the cost of re-prepping those samples is NOT included in the quoted cost. Re-prep is only deemed necessary by the core where we have found evidence of a reagent failure. User request re-preps of individuals samples can be done by hand, but at a higher rate. HTL focuses on reducing price and processing time per sample, so some routine services provided with standard library preparation - such as initial and final quality control for all samples, sample arraying, or automatic re-prep of failed samples - are not built in. These services are available as add ons to the original service request. <br><br>		The BioMicro Center offers high-throuhgput RNA library preparation [[BioMicroCenter:FAQ#STANDARD_versus_HIGH_THROUGHPUT_LIBRARY_PREPARATION\|(HTL-RNA)]] for both standard input and low input samples. Together, these high-throughput methodologies provide a comprehensive set of options to address a diverse range of experiments investigating RNA biology. High-throughput library generation focuses on reducing price and processing time on a per sample basis. To do this, there are several key differences from our standard library preparation service. First, HTL-RNA services have set batch sizes (24 and 96) with fixed price points (i.e. charge for 16 samples will be the as 24 samples; similarly for 80 samples as 96). If under these batch sizes, DO MORE REPLICATES to simultaneously enhance experimental power and reduce price per sample. Some samples may fail and the cost of re-prepping those samples is NOT included in the quoted cost. Re-prep is only deemed necessary by the core where we have found evidence of a reagent failure. User request re-preps of individuals samples can be done by hand, but at a higher rate. HTL focuses on reducing price and processing time per sample, so some routine services provided with standard library preparation - such as initial and final quality control for all samples, sample arraying, or automatic re-prep of failed samples - are not built in. These services are available as add ons to the original service request. <br><br>

			[[Image:DE_HTL_RNA.jpeg\|thumb\|right\|400px\|Increased biological replicates increases power of analysis. (Liu et al. 2014 Bioinformatics 30(3): 301-4)]]

	<b>Reminder: </b> high throughput projects are treated with <b>one condition</b> (PCR cycles, fragmentation time, etc...). They are meant for allowing high replicates of the same samples. High throughput submissions for projects with multiple sub-projects from separate individuals combined on the same plate result in a low success rate due to the different conditions for each sub project. Therefore, htl submissions from multiple projects will tolerate and may expect, failure rates well over 10%.		<b>Reminder: </b> high throughput projects are treated with <b>one condition</b> (PCR cycles, fragmentation time, etc...). They are meant for allowing high replicates of the same samples. High throughput submissions for projects with multiple sub-projects from separate individuals combined on the same plate result in a low success rate due to the different conditions for each sub project. Therefore, htl submissions from multiple projects will tolerate and may expect, failure rates well over 10%.

Hilo1: /* ZapR */

2025-04-30T13:00:26Z

ZapR

← Older revision		Revision as of 13:00, 30 April 2025
Line 221:		Line 221:
	\|		\|
	:SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (ZapR) is used for library preparation of low input degraded RNA samples by generating and amplifying cDNA from total RNA, after which the ribosomal cDNA is depleted through targeted enzymatic degradation using ZapR probes while keeping all other cDNA for library preparation. This method is typically used for low concentration RNA samples of poor quality ([[BioMicroCenter:RIN\|RIN]]<7 or [[BioMicroCenter:RIN\|DV200]]<60%). We have miniaturized the ZapR method to 1/10th the original reaction volume on the Mosquito HV. This method is capable of handling inputs ranging from 250pg to 10ng (or 50ng for FFPE) however, performance for a given sample input is dependent on the quality of the sample itself. Due to the design of the ZapR probes, this method is only available for Human samples.		:SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (ZapR) is used for library preparation of low input degraded RNA samples by generating and amplifying cDNA from total RNA, after which the ribosomal cDNA is depleted through targeted enzymatic degradation using ZapR probes while keeping all other cDNA for library preparation. This method is typically used for low concentration RNA samples of poor quality ([[BioMicroCenter:RIN\|RIN]]<7 or [[BioMicroCenter:RIN\|DV200]]<60%). We have miniaturized the ZapR method to 1/10th the original reaction volume on the Mosquito HV. This method is capable of handling inputs ranging from 250pg to 10ng (or 50ng for FFPE) however, performance for a given sample input is dependent on the quality of the sample itself. Due to the design of the ZapR probes, this method is only available for Human samples.
	[[Image:ZapR_BMC.jpg\|center\|~~250px~~]]		[[Image:ZapR_BMC.jpg\|center\|400px]]
	\|}		\|}

Hilo1: /* ZapR */

2025-04-30T12:59:49Z

ZapR

← Older revision		Revision as of 12:59, 30 April 2025
Line 221:		Line 221:
	\|		\|
	:SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (ZapR) is used for library preparation of low input degraded RNA samples by generating and amplifying cDNA from total RNA, after which the ribosomal cDNA is depleted through targeted enzymatic degradation using ZapR probes while keeping all other cDNA for library preparation. This method is typically used for low concentration RNA samples of poor quality ([[BioMicroCenter:RIN\|RIN]]<7 or [[BioMicroCenter:RIN\|DV200]]<60%). We have miniaturized the ZapR method to 1/10th the original reaction volume on the Mosquito HV. This method is capable of handling inputs ranging from 250pg to 10ng (or 50ng for FFPE) however, performance for a given sample input is dependent on the quality of the sample itself. Due to the design of the ZapR probes, this method is only available for Human samples.		:SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (ZapR) is used for library preparation of low input degraded RNA samples by generating and amplifying cDNA from total RNA, after which the ribosomal cDNA is depleted through targeted enzymatic degradation using ZapR probes while keeping all other cDNA for library preparation. This method is typically used for low concentration RNA samples of poor quality ([[BioMicroCenter:RIN\|RIN]]<7 or [[BioMicroCenter:RIN\|DV200]]<60%). We have miniaturized the ZapR method to 1/10th the original reaction volume on the Mosquito HV. This method is capable of handling inputs ranging from 250pg to 10ng (or 50ng for FFPE) however, performance for a given sample input is dependent on the quality of the sample itself. Due to the design of the ZapR probes, this method is only available for Human samples.
	[[Image:ZapR_BMC.~~gif~~\|center\|250px]]		[[Image:ZapR_BMC.jpg\|center\|250px]]
	\|}		\|}

Hilo1: /* NEB Ultra II Directional RNA with Poly(A) Selection */

2025-04-30T12:57:15Z

NEB Ultra II Directional RNA with Poly(A) Selection

← Older revision		Revision as of 12:57, 30 April 2025
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	:NEBNext Ultra II Directional RNA preparation with poly(A) selection is used to selectively capture mRNA from high-quality total RNA. The selection of mRNA transcripts occurs by hybridization of the poly(A)-tail to magnetic oligo-d(T) beads, which we have miniaturized to 1/6th the original reaction volume using our Tecan Freedom Evo. The purified RNA is then further processed at a 1/10th reaction volume on the Mosquito HV to reliably generate RNA-Seq libraries with inputs ranging from 10ng to 100ng (recommended input of > 50ng). Because this method is reliant on poly(A)-tails during RNA isolation the sample quality significantly impacts performance, as such high-quality samples are an absolute requirement.		:NEBNext Ultra II Directional RNA preparation with poly(A) selection is used to selectively capture mRNA from high-quality total RNA. The selection of mRNA transcripts occurs by hybridization of the poly(A)-tail to magnetic oligo-d(T) beads, which we have miniaturized to 1/6th the original reaction volume using our Tecan Freedom Evo. The purified RNA is then further processed at a 1/10th reaction volume on the Mosquito HV to reliably generate RNA-Seq libraries with inputs ranging from 10ng to 100ng (recommended input of > 50ng). Because this method is reliant on poly(A)-tails during RNA isolation the sample quality significantly impacts performance, as such high-quality samples are an absolute requirement.
	<br>		<br>
	:[[IMAGE:PolyA_HTL.~~png~~ \| 350px]]		:[[IMAGE:PolyA_HTL.jpg \| 350px]]
	\|		\|
	\|}		\|}

Hilo1: /* 3'Digital Gene Expression */

2025-04-30T12:54:43Z

3'Digital Gene Expression

← Older revision		Revision as of 12:54, 30 April 2025
Line 281:		Line 281:
	:Analysis of HT3DGE is not a standard method supported by most open source platforms. We have built pipelines to work with this data, but working closely with the Bioinformatics team is highly recommended.		:Analysis of HT3DGE is not a standard method supported by most open source platforms. We have built pipelines to work with this data, but working closely with the Bioinformatics team is highly recommended.
	\|<br>[[image:DGE_workflow.jpg\|thumb\|right\|275px\|Standard 3'DGE workflow.]]		\|<br>[[image:DGE_workflow.jpg\|thumb\|right\|275px\|Standard 3'DGE workflow.]]
	[[image:HT3DGE_gene_example.jpg\|thumb\|right\|~~275px~~\|Example 3'DGE Data]]<BR><BR>		[[image:HT3DGE_gene_example.jpg\|thumb\|right\|400px\|Example 3'DGE Data]]<BR><BR>
	\|		\|
	\|}		\|}

Hilo1: /* NEB Ultra II Directional RNA with rRNA Depletion */

2025-04-30T12:50:06Z

NEB Ultra II Directional RNA with rRNA Depletion

← Older revision		Revision as of 12:50, 30 April 2025
Line 112:		Line 112:
	:NEBNext Ultra II Directional RNA preparation with ribosomal RNA (rRNA) depletion is used to deplete rRNA by enzymatic degradation using single-stranded DNA probes that target rRNAs, leaving all other RNA species present and available for library preparation. This depletion method is agnostic to input sample quality and is the go-to method if samples don't meet quality requirements for poly(A) selection. We have miniaturized this rRNA depletion method to 1/6th and the following RNA library preparation to 1/10th the original reaction volume on the Mosquito HV. This method is suited for inputs ranging from 5ng to 100ng (recommended input of > 50ng). Due to probe design used in the depletion, this method is currently only available for species where NEB has probes available.		:NEBNext Ultra II Directional RNA preparation with ribosomal RNA (rRNA) depletion is used to deplete rRNA by enzymatic degradation using single-stranded DNA probes that target rRNAs, leaving all other RNA species present and available for library preparation. This depletion method is agnostic to input sample quality and is the go-to method if samples don't meet quality requirements for poly(A) selection. We have miniaturized this rRNA depletion method to 1/6th and the following RNA library preparation to 1/10th the original reaction volume on the Mosquito HV. This method is suited for inputs ranging from 5ng to 100ng (recommended input of > 50ng). Due to probe design used in the depletion, this method is currently only available for species where NEB has probes available.
	<br>		<br>
	:[[IMAGE:Depletion_HTL.~~png~~ \| 350px]]		:[[IMAGE:Depletion_HTL.jpg\|350px]]
	\|		\|
	\|}		\|}

Hilo1: /* 3'Digital Gene Expression */

2025-04-30T12:48:45Z

3'Digital Gene Expression

← Older revision		Revision as of 12:48, 30 April 2025
Line 280:		Line 280:

	:Analysis of HT3DGE is not a standard method supported by most open source platforms. We have built pipelines to work with this data, but working closely with the Bioinformatics team is highly recommended.		:Analysis of HT3DGE is not a standard method supported by most open source platforms. We have built pipelines to work with this data, but working closely with the Bioinformatics team is highly recommended.
	\|<br>[[image:DGE_workflow.~~png~~\|thumb\|right\|275px\|Standard 3'DGE workflow.]]		\|<br>[[image:DGE_workflow.jpg\|thumb\|right\|275px\|Standard 3'DGE workflow.]]
	[[image:HT3DGE_gene_example.~~png~~\|thumb\|right\|275px\|Example 3'DGE Data]]<BR><BR>		[[image:HT3DGE_gene_example.jpg\|thumb\|right\|275px\|Example 3'DGE Data]]<BR><BR>
	\|		\|
	\|}		\|}