title: GSM4876138: C59-D28_scRNA-seq; Homo sapiens; RNA-Seq
library_construction_protocol: Cells were thawed at 37ºC and resuspended in PBS + 0.04% BSA at concentration of 2,000 cells per l. Cell suspensions were submitted to the GTAC sequencing core at Washington University in St. Louis for library preparation and sequencing. In brief, 10,000 cells per sample were loaded on a Chromium Controller (10x Genomics) for single capture. Libraries were prepared using Single Cell 3′ Library & Gel Bead Kit v2 (#120237 10x Genomics) following manufacture's instruction. A single cell emulsion (Gel Bead-In-EMulsions, GEMs) is created by making barcoded cDNA unique to each individual emulsion. Recovery agent was added to break GEM and cDNA was then amplified. A library is produced via end repair, dA-tailing, adaptor ligation, post-ligation cleanup with SPRIselect, and sample index PCR. The quality and concentration of the amplified cDNA was evaluated by Bioanalyzer (Agilent 2100) on a High Sensitivity DNA chip (Agilent, #5065-4401). Only cDNA with average library size 260-620 bp were used for sequencing. Sequencing was performed by Illumina HiSeq2500 with the following read length: 26 bp for Read1, 8 bp for i7 Index, and 98 bp for Read2. We generally acquired ~180 million reads per library (sample).  A species mixing experiment (mouse adipose stem cells and human iPSCs, 1:1 mixture) was also performed prior to running on actual sample to ensure good quality of single cell capture (i.e., cell doublet rate < 5%.). RNA libraries were prepared for sequencing using standard Illumina protocols