GSM6739154: p28floxed-6, 10xRNA; Mus musculus; RNA-Seq
SI-LP cell suspensions were generated as described previously (Luda et al., 2016). Briefly,   after removal of Peyer's patches and fat, tissue was cut longitudinally into 1cm pieces and  incubated in RPMI 1640 (Gibco) containing HEPES (15mM, Gibco),  ethylenediaminetetraacetic acid (EDTA, 2mM) and fetal bovine serum (FBS, Sigma Aldrich,  2%) for 10 min at 37 C with agitation at 370rpm. This step was repeated 2 times with  incubation for 15 min at 37 C. Epithelial cells and debris were removed by filtering through a  nylon filter (250μm) after each wash. Remaining tissue was washed and incubated for 20-25  min in R10 media (RPMI 1640, FBS (10%), HEPES (10mM), sodium pyruvate (1mM),  penicillin (100U/mL), streptomycin (100μg/mL), Gibco) containing 2-ME (50μM, Gibco),  DNase I (30μg/mL, Sigma Aldrich) and Liberase TM (0.3 WuenschU/mL, Sigma Aldrich) on  a magnetic stirring plate at 37°C with 5% CO2. The resulting cell suspensions were filtered   through a cell strainer (100μm, Fischer Scientific) prior to density gradient centrifugation with  40-70% Percoll (GE Healthcare). CD4+ T cells were enriched from SI-LP cell suspensions using the EasySepTM Mouse CD4+ T cell isolation Kit (Stem Cell Technologies) according to the manufacturer's instructions and CD4+TCRαβ+ T cells were sorted using a FACSAria™ II Cell Sorter (BD Bioscience) Isolated CD4+ T cells were subjected to droplet-based scRNA-seq using the Chromium Single Cell 3′ Reagent Kit v3  following the manufacturer's instructions (10x Genomics). The 10x Chromium Controller  generated Gel Bead-In Emulsions (GEMs), where each cell was labeled with a specific  barcode, and each transcript labeled with a unique molecular identifier (UMI). After RT, GEMs  were broken down, barcoded cDNA was purified with Dynabeads MyOne Silane beads  (Thermofisher) and amplified by Polymerase Chain Reaction (PCR). Amplified cDNA was  cleaned up with SPRIselect (Beckman Coulter). Indexed sequencing libraries were constructed  by enzymatic fragmentation, end-repair and A-tailing, before a second and final PCR  amplification using the Chromium i7 Sample Index (10x Genomics), introducing an Illumina  R2 sequence, a unique sample index (allowing multiplex sequencing) and P5/P7 Illumina  sequencing adaptors to each library. Muliplexed libraries were pooled and sequencing was  carried out at the National Genomic Infrastructure (NGI) SNP&SEQ Technology Platform,  Uppsala using an S1 flowcell, the NovaSeq 6000 system and v1.5 sequencing chemistry  (Illumina Inc.).