title: GSM8521718: Fixed RNA profiling of EndoC-betaH1 cells transplanted in vivo; Homo sapiens; RNA-Seq
library_construction_protocol: For ChemPerturb-Seq, we generated twenty-three NLuc-EndoC-betaH1-BC-BFP cell lines, each treated with a different small molecule from our in-house small molecule library  for 48 hours. After 48 hours, NLuc-EndoC-betaH1-BC-BFP cells were trypsinized using 0.25% trypsin-EDTA and pooled. For Fixed RNA profiling, NLuc-EndoC-betaH1-mCherry cells were treated with the LIP cocktail for 48 hours, and 5 million treated or untreated cells were subcutaneously transplanted into male or female SCID-Beige mice. After one week, grafts were removed from the mice and dissociated into single cells using 1 μg/μl of collagenase D for 1 hour at 37C . The mCherry+ cells were purified using either the Becton-Dickinson Influx or Becton-Dickinson Symphony S6 instruments. The cells were then fixed using standard protocols for the Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (10X Genomics). For ChemPerturb-Seq, three different libraries were prepared, and treated and control cells were labeled using the cell multiplexing oligonucleotides from the 3ʹ CellPlex Kit Set A (10X Genomics), specifically, CMO301, CMO302, and CMO303. Libraries were prepared following standard protocols for Chromium Single Cell 3' Reagent Kits with feature barcoding technology for cell multiplexing (10X Genomics) and sequenced on the Illumina NovaSeq 6000 platform. For Fixed RNA profiling, libraries were prepared using the Chromium Fixed RNA Profiling Reagent Kit for Multiplexed Samples (10X Genomics). Samples were multiplexed using the Chromium Fixed RNA Kit, Human Transcriptome, 4 rxns x 4 BC (10X Genomics), and libraries were sequenced using the Illumina NovaSeq 6000 platform.