GSM7786604: SITTH5, sample_7; Homo sapiens; RNA-Seq
Pleura samples were washed three times in PBS, minced with scalpels and digested in RPMI supplemented with collagenase I (5 mg/ml, Life Technologies) and DNase I (100 µg/ml, Sigma) for 2.5 hrs at 37°C. Digested tissue was filtered through a 100μm cell strainer, washed with PBS and centrifuged at 800g. Cell pellets were resuspended first in red blood cell lysis buffer then in RPMI (2% FBS). Dead cells and cellular debris were removed by sorting 7-AAD negative population with BD FACSMelodyTM Cell Sorter (BD Biosciences). Resulting cells were submitted to library preparation. Cells were submitted to 10x Genomics and immediately loaded on 10x3' chip. scRNA-seq libraries were generated using the 10x Chromium platform 3′ v3.1 kit. Library quality was assessed following cDNA synthesis and after completion of the 3' Gene Expression Libraries on the Agilent TapeStation system employing a DNA High Sensitivity D5000 and D1000 chips, respectively(all Agilent). Single cell libraries were sequenced on Illumina NovaSeq 6000 instrument using read 1: 28bp and read 2: 90bp length sequencing and aiming at 20,000 reads/cell.