GSM8828872: 198 s10; Homo sapiens; RNA-Seq
Fresh pinch biopsies were washed twice in DPBS before being coarsely chopped with a scalpel and transferred into 0.5 mL DPBS solution containing 0.1 WU ml-1 Liberase TH (Roche, 5401151001) and 0.5 U ml-1 RQ1 DNase I (Promega, M6101). The samples were incubated at 37 °C in successive 10-minute rounds with gentle vortexing every 2 minutes and trituration every 5 minutes. At the end of each round of incubation, large tissue pieces were spun down in a microcentrifuge and the supernatant containing single cells was passed through a 70-um cell strainer fitted onto a 50-mL conical tube containing 8 mL DPBS + 1% bovine serum albumin (BSA; Sigma-Aldrich, A7906) in DPBS on ice. 0.5 mL fresh dissociation solution was added to the tissue at the beginning of each incubation step. The samples were further filtered through the 0.35 um cell-strainer cap of a FACS tube and single cells were pelleted by centrifugation at 400g for 4 minutes at 4 °C. Samples were washed twice in 1 mL DPBS + 0.04% BSA and finally resuspended in 50 uL DPBS + 0.04% BSA, at which point they were counted in 0.2% trypan blue (Gibco, 15250061) in preparation for 10x Genomics Chromium Next GEM Single Cell 3' v3.1 library preparation. For the frozen BE biopsy from Patient 2, the tissue was quickly thawed at 37 °C and rinsed three times in DPBS + 10% fetal bovine serum (GE, SH30396.03) before being dissociated and filtered as described above. After the final filtering step, dead cells were removed with the Dead Cell Removal Kit (Miltenyi Biotec, 130-090-101) following the manufacturer's protocol. Single-cell suspensions were loaded onto a 10x Chromium Controller for GEM generation of approximately 5,000-10,000 cells for each sample. 10x Genomics Chromium Next GEM Single Cell 3' v3.1 Dual Index library preparation was performed following the manufacturer's protocol. Library quality was confirmed on an Agilent 2100 Bioanalyzer Instrument using the High Sensitivity DNA Kit (Agilent). Libraries were paired-end sequenced on an Illumina NextSeq 550 with 28 cycles for Read 1 and 43 cycles for Read 2, as well as 10 cycles for both indices.