cDNA Library Preparation for scRNA-seq of Human Meniscus (10x Genomics)
molmer, Martin Lotz, Tony Mondala, Steven Head
Abstract
Samples are processed using V2 barcoding chemistry kits of 10x Genomics. For each run, 10,000 cells from individual donors are labeled with distinct oligo-barcoded antibodies (cell hashing), enabling us to pool samples from both conditions on each 10X Genomics run and reduce batch effects. All samples from a given experiment are processed in parallel in the same thermal cycler. The 10x user guide can be found https://assets.ctfassets.net/an68im79xiti/1C16trEdzy1Folq5xbOijE/7e6fb1f504e130bd561d898384da99d9/CG000315_ChromiumNextGEMSingleCell3-_GeneExpression_v3.1_DualIndex__RevB.pdf and is also an attached document.
Attachments
Steps
Single cells are isolated from human knee menicus using doi dx.doi.org/10.17504/protocols.io.n2bvj6k7blk5/v1.
Single cell suspensions are multiplexed and converted to barcoded scRNAseq libraries using the Chromium Single Cell 3’ Library, Gel Bead and Chip Kit (10x Genomics), and the BD™ Hu Single Cell Sample Multiplexing Kit.
Libraries are sequenced on an Illumina NextSeq2000 sequencer targeting 20,000-25,000 reads per cell.
