Editing BioMicroCenter:SingleCell (section)

=== 10X Genomics ===
[[image:10xX.jpg|thumb|right|500px|10x Chromium X]]


FAQs for Users <BR>
'''1) What buffers in the final suspension are compatible with 10X applications?''' 
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[https://kb.10xgenomics.com/hc/en-us/articles/115001937123-What-buffers-can-be-used-for-washing-and-cell-resuspension Buffers/media] for the submitted single cell/nuclei suspension should not contain excessive amounts of EDTA (>0.1 mM) or magnesium (>3 mM) and should be free of surfactants (i.e. Tween-20, SDS etc) and any RNases or DNases.
*1xPBS (calcium free and magnesium free) containing 0.04%  weight/volume BSA (400 µg/ml) is recommended for most general protocols and is considered the standard buffer
*Cell culture media with up to 1% BSA or up to 10% FBS if cells are not viable in standard buffer
*1xPBS (calcium free and magnesium free) containing up to 10% FBS for CMO labelling
*Nuclei also require addition of RNase Inhibitor along with 10X Genomics 1X nuclei buffer before chip loading, instructions for which are included in 10x user guides. If needed, users can collect buffer aliquots from the BMC after submitting a project. Please coordinate with BMC staff for pick-up. <br>
'''2) How many cells are captured in the Assay?'''
<br>Up to 10,000 cells for NextGEM and 20,000 cells for GEM-X can be uniquely barcoded, but this highly depends on cell counts and viability. Dying cells will leak RNA, hence may not be captured efficiently leading to sample failures. We recommend to count cells at the BMC to avoid discrepancies, but can work with users' counts as well. <BR>
'''3) What are the best practices for flow sorting cells? <br> 
10x provides guidance with their tested protocols about pre-sort buffer, collection buffer and FACS best practices [https://kb.10xgenomics.com/hc/en-us/articles/360048826911-What-are-the-best-practices-for-flow-sorting-cells-for-10x-Genomics-assays here.] <br>
'''4) What is the expected size distribution for cDNA?''' <br>
cDNA for 3' and 5' libraries will span between 400 to 9000 base pairs, depending on sample type.
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'''5) How much sequencing per sample is recommended?''' <br>
10x makes several recommendations in their [https://www.10xgenomics.com/support/epi-atac/documentation/steps/sequencing/sequencing-handbook sequencing handbook]. Recommendation numbers vary by sample type, expected CNPs per sample, assay type and general sample quality. The higher the CNPs, the higher the quality, the more likely increased read depth is required. <br>
For more resources, please visit https://kb.10xgenomics.com/hc/en-us <br>
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