GSM9336216: RUUO_UUO_Kidney_2; Mus musculus; OTHER
CD4+ T cells were isolated from the kidneys of RUUO+UUO donor mice in two steps, first with a CD4+ T cell negative isolation kit (Miltenyi) and then with CD4 (L3T4) microbeads (Miltenyi). CD4+ T cells were isolated from the spleens of RUUO+UUO donor mice with CD4 (L3T4) microbeads (Miltenyi). Subsequently FACS-sorting of CD4+ T cells was performed. Total RNA from FACS-sorted CD4+ T cell populations was used for 5' RACE PCR of the TCR-beta chain. For cDNA generation, we introduced a template-switching oligo (TSO, Bio-GGGCUCGGAGAUGUGUAUAAGAGACAGUNNNNUNNNNUNNNNUrGrGrG) that included a unique molecular identifier (UMI). Following reverse transcription, excess TSO was digested using Uracil-DNA Glycosylase (UDG) (New England Biolabs). PCR was performed using the Advantage 2 PCR Kit (Takara) for 20 cycles along with primers specific for the constant region of the Trbc locus (GAAGCCCCTGGCCAAGCACACGAG) as well as a universal forward primer specific for the TSO sequence (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGT). The PCR products were purified with AMPure XP beads (Beckman Coulter) and reactions were split into technical replicates for the successive extension PCR (15 cycles) using the Advantage 2 PCR Kit (Takara) and TSO (GTCTCGTGGGCTCGGAGATGTG) and another Tcrb-specific primer (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTGATGGCTCAAACAAGGAG). Barcoding was performed in a third PCR using IDT for Illumina DNA/RNA UD Indexes (10 cycles). Libraries were purified using AMPure XP beads (Beckman Coulter), concentrations were measured with the Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific) and fragment size profiles were assessed using the High Sensitivity D1000 or D5000 ScreenTape Assay (Agilent). Sequencing (paired-end 300bp) was performed on the Illumina NextSeq 2000 system.