GSM6995653: spleenTreg_3-5_GvHD_Tcra_R1; Mus musculus; OTHER
Total cellular RNA was isolated from Treg (freshly isolated, in vitro expanded, or re-isolated after BMT) using the RNeasy Mini Kit or the RNeasy Micro Kit (Qiagen). Following the manufacturer's recommendations, samples originating from 500 to 500k cells were processed with the Micro Kit whereas the Mini Kit was applied to all samples exceeding 500k cells. RNA concentration and quality were simultaneously assessed using RNA Screentape Analysis or High Sensitivity RNA Screentape Analysis (Agilent), depending on the expected yield. Total cellular RNA was obtained as described in section “RNA-seq library preparation”. TCR repertoire sequencing libraries for Illumina sequencing were prepared on the basis of {Mamedov, 2013 #7} and the protocol was adapted, as follows. For the generation of 5'RACE-ready cDNA total RNA ranging from 10 - 200 ng (low input) or 200 - 1000 ng (high input) in a total of 10 µl RNAse-free water was combined with 1 µL of 12 µM or 25 µM 5'-CDS Primer A in a total volume of 11 µL, respectively. Reactions were incubated at 72°C for 3 min followed by 42°C for 2 min for poly-dT primer annealing. Next, 1 µL of 10 µM (low input) or 50 µM (high input) TCR_UMI_Smarter template-switching oligo (TSO) was added at room temperature. RT Buffer Mix (5x First-Strand-Buffer Mix, 20mM dNTP, 100 mM DTT) was freshly prepared on ice before it was supplemented with RNaseOUT™ Recombinant Ribonuclease Inhibitor (40 U/µL) (Thermo Fisher Scientific) and SMARTScribe Reverse Transcriptase (100 U/µL) (Takara) at room temperature. A volume of 8 µL of the resulting reverse transcription master mix was added to each reaction. After homogenization, the 20 µL reactions were incubated at 42°C for 90 min followed by 70°C for 10 min and put on hold at 10°C in a hot-lid thermal cycler. Following reverse transcription, excess TSO was digested using Uracil-DNA Glycosylase (UDG) (New England Biolabs) by adding 4 µL of UDG master mix adjusted for TSO input amount and successive incubation at 37°C for 60 min. The master mix for PCR1 was prepared on ice using the Advantage 2 PCR Kit (Takara) along with primers specific for the constant regions of the Trac and Trbc loci as well as a universal forward primer specific for a binding sequence introduced via the TSO. 24 µL of RACE-ready cDNA were combined with 26 µL of PCR1 master mix and subjected to a two-step PCR program (95°C, 1min; 95°C, 30 s & 68°C, 70 s (x times); 68°C, 7 min; 8°C, hold). Depending on the RNA input amount, PCR cycle numbers ranged from 16 to 28. The 50 µL PCR product was purified with 27.5 µL of AMPure XP beads (Beckman Coulter) according to the manufacturer's instructions. After elution in 23.5 µL of Monarch® DNA Elution Buffer (New England Biolabs), reactions were split into 10 µL technical replicates for successive extension PCR (PCR2). PCR2 master mix was assembled using the Advantage 2 PCR Kit (Takara) and gene-specific nested primers (Trac, Trbc loci) introducing Illumina Nextera XT i5 indices (S501, S502, S503, S508, S517, S520, S507), distinguishing Tcra from Tcrb libraries within each reaction. After addition of 36 µl PCR2 master mix, 4 µL of Nextera XT Index Kit v2 (Illumina) adapters were used for indexing libraries individually. Libraries were amplified using the same program as in PCR1 with 18 PCR cycles. Library purification was carried out using  AMPure XP beads (Beckman Coulter) at a volume ratio of 0.8 according to the manufacturer's instructions. After final elution in 17 µL of Monarch® DNA Elution Buffer (New England Biolabs), concentration was measured with the Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific) and fragment size profile was assessed using the High Sensitivity D1000 or D5000 ScreenTape Assay (Agilent). Libraries were stored at -20°C until sequencing (paired-end 300bp) on the Illumina MiSeq system. Oligonucleotide sequences are summarized in Supplemental Table 3, Libraries are summarized in Supplemental Tables 4-7. poly-dT-primed, gene-specifically enriched TRA/TRB library preparation