GSM4100728: PDAC-G ST1; 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).