BioMicroCenter:RNA LIB - Revision history

172.18.0.1: /* Clontech SMARTseq Low-Input */

2025-05-02T14:52:03Z

Clontech SMARTseq Low-Input

← Older revision		Revision as of 14:52, 2 May 2025
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	For samples with less then 50ng of input, the BioMicro Center utilizes the [http://www.clontech.com/US/Products/cDNA_Synthesis_and_Library_Construction/cDNA_Synthesis_Kits/Ultra_Low_Input_RNA_cDNA_Synthesis Clontech SMARTseq v4 system]. This system differs from the TruSeq chemistry in that it begins with cDNA generation using polyT priming followed by strand switching oligos. The use of polyT priming requires the RNA to be of high quality. Full length double-stranded cDNAs are generated and amplified by PCR. These cDNAs ~~are then~~ prepared into ~~Illumina~~ libraries using the NexteraXT chemistry from Illumina. Data from this system is of similar quality to samples created with Illumina TruSeq chemistry but is not stranded. Single samples can be prepared by hand. [[BioMicroCenter:RNA_HTL\|Batches of 24, 96 or 384 samples]] can be prepared using the older SMARTseq v2 chemistry on the Mosquito HV resulting in significantly lower costs/sample.		For samples with less then 50ng of input, the BioMicro Center utilizes the [http://www.clontech.com/US/Products/cDNA_Synthesis_and_Library_Construction/cDNA_Synthesis_Kits/Ultra_Low_Input_RNA_cDNA_Synthesis Clontech SMARTseq v4 system]. This system differs from the TruSeq chemistry in that it begins with cDNA generation using polyT priming followed by strand switching oligos. The use of polyT priming requires the RNA to be of high quality. Full length double-stranded cDNAs are generated and amplified by PCR. These cDNAs can be prepared into NGS short-read libraries using the NexteraXT chemistry from Illumina. Data from this system is of similar quality to samples created with Illumina TruSeq chemistry but is not stranded. Single samples can be prepared by hand. [[BioMicroCenter:RNA_HTL\|Batches of 24, 96 or 384 samples]] can be prepared using the older SMARTseq v2 chemistry on the Mosquito HV resulting in significantly lower costs/sample.

	\|}		\|}

172.18.0.1 at 14:49, 2 May 2025

2025-05-02T14:49:46Z

← Older revision		Revision as of 14:49, 2 May 2025
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			<!-- commenting Kapa out
	==[https://sequencing.roche.com/en-us/products-solutions/by-category/library-preparation/rna-library-preparation/kapa-mrna-hyperprep-kits.html Kapa mRNA Hyperprep] ==		==[https://sequencing.roche.com/en-us/products-solutions/by-category/library-preparation/rna-library-preparation/kapa-mrna-hyperprep-kits.html Kapa mRNA Hyperprep] ==
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			end of kapa -->
	== [http://www.clontech.com/US/Products/cDNA_Synthesis_and_Library_Construction/cDNA_Synthesis_Kits/Ultra_Low_Input_RNA_cDNA_Synthesis Clontech SMARTseq Low-Input] ==		== [http://www.clontech.com/US/Products/cDNA_Synthesis_and_Library_Construction/cDNA_Synthesis_Kits/Ultra_Low_Input_RNA_cDNA_Synthesis Clontech SMARTseq Low-Input] ==
	{\|		{\|

Hilo1: /* Clontech SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian -- aka Clontech ZapR */

2025-04-30T13:09:59Z

Clontech SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian -- aka Clontech ZapR

← Older revision		Revision as of 13:09, 30 April 2025
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	For samples with less then 100ng of input and restricted input amounts, our kit of choice is the Clontech SMARTer Stranded Total RNAseq Kit - Pico Input -- or more simply, Clontech ZapR . This kit utilizes the same template switching as the v4 kit but uses random primers on fragmented RNA. The key is the ZapR enzyme which is used post library production to, in a targeted manner, cause breaks in Illumina library molecules that contain rRNA reads. These breaks make the rRNA containing molecules unreadable. ~~Currently this~~ chemistry ~~is only available~~ as ~~single samples but we are working to adapt it to the Mosquito HV system~~. <BR><BR>		For samples with less then 100ng of input and restricted input amounts, our kit of choice is the Clontech SMARTer Stranded Total RNAseq Kit - Pico Input -- or more simply, Clontech ZapR . This kit utilizes the same template switching as the v4 kit but uses random primers on fragmented RNA. The key is the ZapR enzyme which is used post library production to, in a targeted manner, cause breaks in Illumina library molecules that contain rRNA reads. These breaks make the rRNA containing molecules unreadable. This chemistry can be utilized in HTL format as well. <BR><BR>

	In analyzing data from this kit, we have observed that the first few nucleotides from many reads appear to have a very high mismatch rate, particularly from low input samples or samples that possibly may not be as clean as desired. We believe this is a results of the template switching and random priming. A 5nt trim from the 5'end of the read can significantly improve data quality.		In analyzing data from this kit, we have observed that the first few nucleotides from many reads appear to have a very high mismatch rate, particularly from low input samples or samples that possibly may not be as clean as desired. We believe this is a results of the template switching and random priming. A 5nt trim from the 5'end of the read can significantly improve data quality.
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	\|}		\|}

	== Additional Chemistries Available in the BioMicro Center ==		== Additional Chemistries Available in the BioMicro Center ==

	=== [[BioMicroCenter:PippinPrep\|Size Selection]] ===		=== [[BioMicroCenter:PippinPrep\|Size Selection]] ===
	For some applications of RNAseq, such as splice choice determination, having a precise knowledge of the insert size is critical. While a standard SPRI clean does provide some size selection (typically restricting fragments to between 150 and 350bp), this can be too wide for some methodologies. In these cases, after libraries are amplified, they can be run on the [[BioMicroCenter:PippinPrep\|Sage BluePippin]] (either singly or pooled). Here the size distribution can be much tighter, with most of the DNA fragments being within a 50nt range.		For some applications of RNAseq, such as splice choice determination, having a precise knowledge of the insert size is critical. While a standard SPRI clean does provide some size selection (typically restricting fragments to between 150 and 350bp), this can be too wide for some methodologies. In these cases, after libraries are amplified, they can be run on the [[BioMicroCenter:PippinPrep\|Sage BluePippin]] (either singly or pooled). Here the size distribution can be much tighter, with most of the DNA fragments being within a 50nt range.

Hilo1: /* Size Selection */

2025-04-30T13:08:53Z

Size Selection

← Older revision		Revision as of 13:08, 30 April 2025
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	=== [[BioMicroCenter:PippinPrep\|Size Selection]] ===		=== [[BioMicroCenter:PippinPrep\|Size Selection]] ===
	For some applications of RNAseq, such as splice choice determination, having a precise knowledge of the insert size is critical. While ~~the [[BioMicroCenter:~~SPRI~~-Works\|SPRIworks]]~~ does provide some size selection (typically restricting fragments to between 150 and 350bp), this can be too wide for some methodologies. In these cases, after libraries are amplified, they can be run on the [[BioMicroCenter:PippinPrep\|Sage BluePippin]] (either singly or pooled). Here the size distribution can be much tighter, with most of the DNA fragments being within a 50nt range.		For some applications of RNAseq, such as splice choice determination, having a precise knowledge of the insert size is critical. While a standard SPRI clean does provide some size selection (typically restricting fragments to between 150 and 350bp), this can be too wide for some methodologies. In these cases, after libraries are amplified, they can be run on the [[BioMicroCenter:PippinPrep\|Sage BluePippin]] (either singly or pooled). Here the size distribution can be much tighter, with most of the DNA fragments being within a 50nt range.

Hilo1 at 13:07, 30 April 2025

2025-04-30T13:07:41Z

← Older revision		Revision as of 13:07, 30 April 2025
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	\| >10ng \|\| RIN:>8.0 \|\| [[BioMicroCenter:RNA_LIB#NEB_Ultra_II_Directional_RNA_with_Poly(A)_Selection\|NEB Poly A]]		\| >10ng \|\| RIN:>8.0 \|\| [[BioMicroCenter:RNA_LIB#NEB_Ultra_II_Directional_RNA_with_Poly(A)_Selection\|NEB Poly A]]
	\|-		\|-
	\| >50ng \|\| DV200>0.2 \|\| ~~[[BioMicroCenter:RNA_LIB]]~~ NEB ~~or Lexogen~~ ribosomal depletion followed by NEB UltraII prep.		\| >50ng \|\| DV200>0.2 \|\| NEB ribosomal depletion followed by NEB UltraII prep.
	\|-		\|-
	\| 10pg-25ng \|\| RIN:9.0 \|\| [[BioMicroCenter:RNA_LIB#Clontech_SMARTseq_Low-Input\|Clontech SMARTer v4]]		\| 10pg-25ng \|\| RIN:9.0 \|\| [[BioMicroCenter:RNA_LIB#Clontech_SMARTseq_Low-Input\|Clontech SMARTer v4]]

Hilo1 at 13:07, 30 April 2025

2025-04-30T13:07:07Z

← Older revision		Revision as of 13:07, 30 April 2025
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	! Method Recommended		! Method Recommended
	\|-		\|-
	\| >10ng \|\| RIN:>8.0 \|\| [[BioMicroCenter:RNA_LIB#NEB_Ultra_II_Directional_RNA_with_Poly(A)_Selection\|NEB Poly A~~]] or [[BioMicroCenter:RNA_LIB#Kapa_mRNA_Hyperprep\|Kapa Hyperprep~~]]		\| >10ng \|\| RIN:>8.0 \|\| [[BioMicroCenter:RNA_LIB#NEB_Ultra_II_Directional_RNA_with_Poly(A)_Selection\|NEB Poly A]]
	\|-		\|-
	\| >50ng \|\| DV200>0.2 \|\| [[BioMicroCenter:RNA_LIB]] NEB or Lexogen ribosomal depletion followed by NEB UltraII prep.		\| >50ng \|\| DV200>0.2 \|\| [[BioMicroCenter:RNA_LIB]] NEB or Lexogen ribosomal depletion followed by NEB UltraII prep.

Hilo1 at 18:19, 25 April 2025

2025-04-25T18:19:37Z

← Older revision		Revision as of 18:19, 25 April 2025
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	{{BioMicroCenter}}		{{BioMicroCenter}}

	[[Image:RNA-Seq_pres.jpg\|thumb\|300px\|right\|Wang ET, et al. Nat Rev Genet 2009]]		[[Image:RNA-Seq_pres.jpg\|thumb\|300px\|right\|Wang Z, et al. Nat Rev Genet 2009]]
	The BioMicro Center supports a broad variety of standard library preparation methods for RNAseq. The choice of method is highly dependent on the type of input, the amount of input RNA available, and the quality of the input RNA. The key in all RNAseq methods is the avoidance of ribosomal RNA, which would dominate the library preparation. Below is a summary of the methods we utilize routinely in the core. For High-Throughput RNA library preparation, please see our page for [[BioMicroCenter:RNA_HTL\|methods designed specifically for large sample batches.]]		The BioMicro Center supports a broad variety of standard library preparation methods for RNAseq. The choice of method is highly dependent on the type of input, the amount of input RNA available, and the quality of the input RNA. The key in all RNAseq methods is the avoidance of ribosomal RNA, which would dominate the library preparation. Below is a summary of the methods we utilize routinely in the core. For High-Throughput RNA library preparation, please see our page for [[BioMicroCenter:RNA_HTL\|methods designed specifically for large sample batches.]]

Hilo1 at 18:18, 25 April 2025

2025-04-25T18:18:12Z

← Older revision		Revision as of 18:18, 25 April 2025
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	{{BioMicroCenter}}		{{BioMicroCenter}}

	[[Image:RNA-Seq_pres.jpg\|thumb\|300px\|right\|Wang ET, et al. ~~Nature 2008~~]]		[[Image:RNA-Seq_pres.jpg\|thumb\|300px\|right\|Wang ET, et al. Nat Rev Genet 2009]]
	The BioMicro Center supports a broad variety of standard library preparation methods for RNAseq. The choice of method is highly dependent on the type of input, the amount of input RNA available, and the quality of the input RNA. The key in all RNAseq methods is the avoidance of ribosomal RNA, which would dominate the library preparation. Below is a summary of the methods we utilize routinely in the core. For High-Throughput RNA library preparation, please see our page for [[BioMicroCenter:RNA_HTL\|methods designed specifically for large sample batches.]]		The BioMicro Center supports a broad variety of standard library preparation methods for RNAseq. The choice of method is highly dependent on the type of input, the amount of input RNA available, and the quality of the input RNA. The key in all RNAseq methods is the avoidance of ribosomal RNA, which would dominate the library preparation. Below is a summary of the methods we utilize routinely in the core. For High-Throughput RNA library preparation, please see our page for [[BioMicroCenter:RNA_HTL\|methods designed specifically for large sample batches.]]

Hilo1: /* Clontech SMARTseq Low-Input */

2025-04-25T18:16:20Z

Clontech SMARTseq Low-Input

← Older revision		Revision as of 18:16, 25 April 2025
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	For samples with less then 50ng of input, the BioMicro Center utilizes the [http://www.clontech.com/US/Products/cDNA_Synthesis_and_Library_Construction/cDNA_Synthesis_Kits/Ultra_Low_Input_RNA_cDNA_Synthesis Clontech SMARTseq v4 system]. This system differs from the TruSeq chemistry in that it begins with cDNA generation using polyT priming followed by strand switching oligos. The use of polyT priming requires the RNA to be of high quality. Full length double-stranded cDNAs are generated and amplified by PCR. These cDNAs are then prepared into Illumina libraries using the NexteraXT chemistry from Illumina. Data from this system is of similar quality to samples created with Illumina TruSeq chemistry but is not stranded. Single samples can be prepared by hand. [[BioMicroCenter:RNA_HTL\|Batches of 24, 96 or 384 samples]] can be prepared using the older SMARTseq v2 chemistry on the Mosquito HV resulting in significantly lower costs/sample.		For samples with less then 50ng of input, the BioMicro Center utilizes the [http://www.clontech.com/US/Products/cDNA_Synthesis_and_Library_Construction/cDNA_Synthesis_Kits/Ultra_Low_Input_RNA_cDNA_Synthesis Clontech SMARTseq v4 system]. This system differs from the TruSeq chemistry in that it begins with cDNA generation using polyT priming followed by strand switching oligos. The use of polyT priming requires the RNA to be of high quality. Full length double-stranded cDNAs are generated and amplified by PCR. These cDNAs are then prepared into Illumina libraries using the NexteraXT chemistry from Illumina. Data from this system is of similar quality to samples created with Illumina TruSeq chemistry but is not stranded. Single samples can be prepared by hand. [[BioMicroCenter:RNA_HTL\|Batches of 24, 96 or 384 samples]] can be prepared using the older SMARTseq v2 chemistry on the Mosquito HV resulting in significantly lower costs/sample.
	\|
	~~[[Image:BMC_ClontechChemistry.png\|thumb\|right\|200px\|Clontech system.<BR>Image from Clonetech]]~~
	\|}		\|}

Hilo1: /* Kapa mRNA Hyperprep */

2025-04-25T18:15:42Z

Kapa mRNA Hyperprep

← Older revision		Revision as of 18:15, 25 April 2025
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	This workflow is very similar to Illumina's TruSeq chemistry at lower cost and is streamlined for automation. This chemistry uses polyT beads to isolate the mRNA from the rRNA and tRNA. The use of these beads requires that the RNA be of very high quality or only the 3' end of transcripts will be isolated. Purified mRNA is then fragmented with metal and random priming is used to convert the sample to cDNA. Once double-stranded cDNA is generated, LMPCR is performed to create the indexed Illumina library. The BioMicro Center offers mRNA HyperPrep as a single sample reaction or in [[BioMicroCenter:RNA_HTL\|batches of 24 done on the TecanEvo 150s.]]		This workflow is very similar to Illumina's TruSeq chemistry at lower cost and is streamlined for automation. This chemistry uses polyT beads to isolate the mRNA from the rRNA and tRNA. The use of these beads requires that the RNA be of very high quality or only the 3' end of transcripts will be isolated. Purified mRNA is then fragmented with metal and random priming is used to convert the sample to cDNA. Once double-stranded cDNA is generated, LMPCR is performed to create the indexed Illumina library. The BioMicro Center offers mRNA HyperPrep as a single sample reaction or in [[BioMicroCenter:RNA_HTL\|batches of 24 done on the TecanEvo 150s.]]
	\|		\|
	{\|
	~~[[Image:BMC_RNAseqMethod.png\|thumb\|right\|200px\|TruSeq Chemistry]]~~
	\|-
	~~[[Image:BMC_IlluminaRNAseq.png\|thumb\|right\|300px\|Sample Data]]~~
	\|}
	\|}		\|}