GSM6214746: PDX 10x scRNA-seq; Homo sapiens; Mus musculus; RNA-Seq
For inDrop single-cell RNA-Seq of patient tumor samples, cell suspension was generated from minced tumor tissue using human tumor dissociation kit (Miltenyi) prior to droplet encapsulation and individual barcoding of cells using the inDrop platform. Cell lysis was performed in droplets to release RNA, and reverse transcription and barcoding took place in droplets at 50C for 2h. For 10x Genomics spatial transcriptomics of patient tumor samples, 10 µm cryosections were mounted onto spatially barcoded arrays where reverse transcription of cellular transcripts took place in situ after tissue permeabilisation for 12 minutes. For 10x Genomics single-cell RNA-Seq of mouse tumors, cell suspension was generated from minced tumor tissue using the mouse tumor dissociation kit (Miltenyi) enzymes D and R prior to droplet encapsulation and individual barcoding of cells using the 10x Genomics platform. For inDrop single-cell RNA-seq library construction, we followed the inDrop library construction protocol detailed by Klein et al,  Cell 2015, with minor changes. Briefly, RNA was reverse transcribed (RT) with SuperScript III (Invitrogen) in droplets. Droplet emulsions were broken, and post-RT material underwent second strand syntehsis and in vitro transcription using the T7 High Yield Enzyme mix (New England BiolaBS). RNA was fragmented for 105 seconds with 1X fragmentation reagent (1CellBio) prior to RT with random hexamers, eliminating the need for an adaptor ligation step. Cycles required for final library amplification was assessed by quantitative PCR (qPCR) with KAPA HiFi Hot Start PCR Mix (KAPA Biosystems) and EvaGreen dye (Biotium). Final libraries were amplified with KAPA HiFi Hot Start PCR mix for 9 to 13 cycles. inDrop library size assessed on a DNA BioAnalyzer chip following the manufacturer's instructions (Agilent).   For 10x Genomics spatial transcriptomic and single-cell RNA-Seq library construction, we followed the manufacturer's instructions.