GSM3405529: PDAC-A inDrop5; Homo sapiens; RNA-Seq
For single-cell RNA-seq (inDrop platform), cell suspension was generated by incubating minced pancreatic cancer tissue with trypsin for 15' at 37C 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 spatial transcriptomics of pancreatic cancer tissue, 10 µm cryosections were mounted onto spatially barcoded arrays where reverse transcription of cellular transcripts took place in situ after tissue permeabilisation. For 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 spatial transcriptomics (ST) library construction, we followed the previously published ST protocol from Stahl et al. Science 2016, with minor changes. Briefly, after brightfield imaging of stained tissue, tissue was permeabilized with collagenase and 0.1% pepsin prior to an overnight RT step with SuperScript III on the ST slide. Tissue was digested away with proteinase K and 1% 2-mercaptoethanol prior to cleavage of probes from the slide surface with USER enzyme. Second strand synthesis was performed with DNA Pol I and RNase H (New england biolabs) followed by an in vitro transcription amplification step with the MEGAScript T7 kit (Thermofisher). Amplified RNA then underwent a second RT with random hexamers and SuperScript II (Invitrogen), 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).