title: GSM7794794: Ocab(fl/fl) - 1; Mus musculus; RNA-Seq
library_construction_protocol: At remission timepoint, brain and spinal cord cells were FACS sorted for CD3e+ cells. The Chromium Next GEM Single Cell 5' Reagent kits v2 (PN-1000265) was used to barcode individual cells with oligonucleotides containing an Illumina R1 sequence, a 16 nt 10x Barcode, a 10 nt Unique Molecular Identifier (UMI), and  a 13 nt template switch oligo (TSO) according to the manufacture.  The following protocol based on 10x Genomics user guide (CG000331) was performed by High-Throughput Genomics Shared Resource at Huntsman Cancer Institute, University of Utah.  Filtered CD3e+ cells  with 10x Gel Beads and reverse transcription reagents were loaded to Chromium Single Cell Chip K (PN-1000287) to form Gel-Bead-In EMulsions (GEMs), the nano-droplets.  Within individual GEMs, barcoded cDNA generated from captured poly-adenylated mRNA was synthesized by reverse transcription (RT) with a master mix containing reverse transcription reagents and poly d(T) RT primer at the setting of 53°C for 45 min followed by 85°C for 5 min.  Subsequent fragmentation, end repair and A-tailing, adaptor ligation and sample indexing with dual index (PN-1000215) were performed in bulk according to the user guide.  The resulting barcoded libraries were qualified using Agilent D1000 ScreenTape on Agilent Technology 4150 TapeStation system and quantified by quantification PCR using KAPA Biosystems Library Quantification Kit for Illumina Platforms (KK4842).  Multiple libraries were then normalized, pooled and sequenced on NovaSeq 6000 with 150x150 paired-end mode