GSM4998195: SCLC1; Homo sapiens; RNA-Seq
Resected tumor was transported in MACS Tissue Storage Solution (Miltenyi Biotec) on ice immediately after surgical procurement. A small fragment was fixed in 4% paraformaldehyde (Solarbio) at 4 °C overnight and then paraffin embedded for immunohistochemical analysis and whole-exome sequencing (WES). Remaining tumor was minced into tiny cubes (<1 mm3), and enzymatically digested with MACS tumor dissociation kit (Miltenyi Biotec) for 60 min on a rotor at 37 °C, according to the manufacturer's instructions. After digestion, the cells were centrifuged at 400 g at 4 °C for 5 min, and then resuspended in 100U/ml DNase I (Sigma-Aldrich Cat# D4527) and incubated at 37 °C for 15 min. After filtering and centrifuge, the pellet was suspended in red blood cell lysis buffer (Solarbio) and incubated on ice for 2 min to lyse red blood cells. And the solution diluted with HBSS then centrifuged (600 g, 5 minutes, 4°C). The supernatant was removed, and the cells resuspended in appropriate solution for further analysis. The protoplast suspension was loaded into Chromium microfluidic chips with 30 v3 chemistry and barcoded with a 10× Chromium Controller (10X Genomics). RNA from the barcoded cells was subsequently reverse-transcribed and sequencing libraries constructed with reagents from a Chromium Single Cell 30 v3 reagent kit (10X Genomics) according to the manufacturer's instructions. Sequencing was performed with Illumina nova6000 according to the manufacturer's instructions (Illumina).