GSM8752776: a199 s8; 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. Mitochondrial transcripts were enriched from the intermediate 10x cDNA libraries following the MAESTER protocol22. Briefly, previously amplified full-length cDNA was further amplified over six cycles in 12 separate PCR reactions containing primers that together spanned the mitochondrial transcriptome; 5-20 ng input cDNA was used in each reaction. PCR products for each sample were pooled and purified using Ampure XP (Beckman Coulter, A63881). Sequencing primer binding sites, adapters, and dual indices were added to the resulting cDNA over another six cycles of PCR and again purified using Ampure XP. Final MAESTER libraries were in the expected range of 2-100 ng ul-1 for concentration and fragment sizes of 300-1,500 bp. Libraries were sequenced on an Illumina NextSeq 550 with a v2.5 300 cycle kit, allocating 28 cycles for Read 1 and 270 cycles for Read 2, as well as eight cycles for both indices; a custom index 2 primer was used (10x-Ci5P).