GSM4060089: Bisulfite-seq_Donor 6.5TCR T cells which were not converted to Tregs, rep2; Mus musculus; OTHER
T cell subsets were sorted directly into FACS buffer and then lysed with buffer ATL and proteinase K (Qiagen QIAamp DNA Micro Kit #56304) for one hour at 56°C. Buffer ATL and AL with carrier RNA were added and sample stored at -80°C until further processing. To isolate genomic DNA, samples were heated to 90°C for 30 minutes followed by column-based DNA isolation according to manufacturer's protocol (Qiagen QIAamp DNA Micro Kit #56304). DNA was eluted in 40 µl buffer AE and sodium bisulfite conversion was performed (Qiagen EpiTect Plus DNA Bisulfite Kit #59124). Bisulfite-converted DNA was used to amplify regions of interest56 with the following PCR conditions: 4 µl bisulfite-converted DNA, 0.5 µl forward primer (10 µM), 0.5 µl reverse primer (10 µM), 5 µl 2X PCR buffer, 0.1 µl dNTPs and 0.1 µl hot-start Taq polymerase (PCR buffer, dNTPs and Taq from: ZymoTaq DNA Polymerase Kit #E2002). To detect Foxp3 methylation at the Treg-specific demethylated region, we used a forward primer TGGGTTTTTTTGGTATTTAAGAAAG and a reverse primer AAAAAACAAATAATCTACCCCACAA. To detect Il2ra gene methylation, we used a forward primer TTTTAGAGTTAGAAGATAGAAGGTATGGAA and a reverse primer TCCCAATACTTAACAAAACCACATAT. For Ikzf2 gene methylation, we used a forward primer AGGATGGTTTTTATTGAAGGTGAT and a reverse primer ATACACACCAAACAAACACTACACC. For Ctla4 gene methylation, we used a forward primer TGGTGTTGGTTAGTAGTTATGGTGT and a reverse primer AAATTCCACCTTACAAAAATACAATC. For Tnfrsf18 gene methylation, we used a forward primer GAGGTGTAGTTGTTAGTTGAGGATGT and a reverse primer AACCCCTACTCTCACCAAAAATATAA. PCR was initiated with 95°C for 10 minutes, followed by 40 cycles with denaturation (95°C-30sec), annealing (56°C-30sec) and elongation (72°C-30sec) completed with a final elongation step (72°C for 7 minutes). PCR products were combined, pre-purified with AMPure XP beads (Beckman Coulter #463881) and indexed for Illumina-based sequencing (NEBNext Ultra II DNA Library Prep Kit for Illumina #E7645S with NEBNext Multiplex Oligos for Illumina #E7335S). Samples were pooled in an equimolar ratio and supplemented with an unindexed PhiX spike-in (50% spike-in, Illumina FC-110-3001). Sequencing was performed on an Illumina MiSeq with a reagent nano kit v2 and 500 cycles of PCR.
GEO Accession
GSM4060089