title: GSM8217900: CKI_HTO_S1,S2,S3,S4,S5; Mus musculus; RNA-Seq
library_construction_protocol: Hash-tagged and Total-seq-C cocktail labelled cells wee sorted for live CD45+ and equal cell numbers of 5 samples from each treatment group were pooled into one sample per treatment group. Total RNA was extracted and quantified prior to sequencing. GEM generation, cDNA synthesis, cDNA amplification, and library preparation of 6,700-11,400 cells proceeded using the Chromium Next GEM Single Cell 5' Kit v2 (10X Genomics PN 1000263) according to the manufacturer's protocol. cDNA amplification included 13 cycles and 9.4-43 ng of the material was used to prepare sequencing libraries with 14-16 cycles of PCR. Indexed libraries were pooled equimolar and sequenced on a NovaSeq 6000 in a PE28/88 run using the NovaSeq 6000 S1 or S2 Reagent Kit (100 cycles) (Illumina).  An average of 44 thousand reads was generated per cell. Amplification products generated using the methods described above included both cDNA and feature barcodes tagged with cell barcodes and unique molecular identifiers. Smaller feature barcode fragments were separated from longer amplified cDNA using a 0.6X cleanup using aMPure XP beads (Beckman Coulter catalog # A63882). Libraries were constructed using the 5' Feature Barcode Kit (10X Genomics PN 1000256) according to the manufacturer's protocol with 8 cycles of PCR. Indexed libraries were pooled equimolar and sequenced on a NovaSeq 6000 in a PE28/88 run using the NovaSeq 6000 S1 Reagent Kit (100 cycles) (Illumina). An average of 58 million reads was generated per sample.