GSM8586650: Control TINs, replicate 1, plate 05, scRNAseq; Mus musculus; RNA-Seq
For isolation of single tumor cells, tumors were mechanically dissociated with scissors and digested by incubation for 30 min at 37°C with collagenase at 1 mg/mL in HBSS. Cells were then isolated by vigorous pipetting of the tumor fragments and passage through a 70-μm cell strainer. For analysis of CCA tumors, erythrocytes were lysed with hemolysis buffer (0.14 M NH4Cl and 0.01 M Tris-HCl at pH 7.5), and the remaining cells were stained with antibodies against CD45.2 (clone 104, BioLegend), CD11b (clone M1/70, BioLegend), Ly6G (clone 1A8, BioLegend). Cell sorting was performed with a FACS Aria II instrument fitted with a 100-μm nozzle. After gating for forward scatter and side scatter and exclusion of doublets, the 7AAD–CD45+CD11b+Ly6G+ fraction was collected as neutrophils. For single-cell RNA sequencing (scRNA-seq) library preparation, single TINs were processed and barcoded with the use of the CEL-seq2 technique as performed with minor modifications. In brief, each single cell was directly sorted into a well of a 96-well PCR plate containing primer mix including reverse transcription (RT) primers with unique molecular identifiers (UMI) and cell barcodes, deoxynucleoside triphosphates, RNase inhibitors, and 0.3% Nonidet P-40. The cells were lysed by incubation at 65°C for 5 minutes and then subjected to room temperature with SuperScript III Reverse Transcriptase. Samples from each plate were pooled into a single library, from which cDNAs were purified with the use of Agencourt AMPure XP Beads and subjected to second-strand synthesis. The resultant libraries were again bead-purified and then used as templates for in vitro transcription with a MEGAscript T7 Transcription Kit. The amplified RNAs were subjected to RT followed by PCR amplification (20 cycles) with Illumina TruSeq Small RNA primers to generate the final cDNA libraries, which were subjected to paired-end sequencing with an Illumina NovaSeq6000 instrument.