title: 546421cbf94adc40
design: The experimental protocol used for Mint-ChIP data generation was similar to Schuetter et al. Briefly, cryopreserved PBMCs were thawed on ice, and viability was assessed using Trypan blue and a hemocytometer. Between 50,000 and 100,000 viable cells from each sample were lysed for 1 hour and 4C. Nuclei were digested with 300 units of micrococcal nuclease (MNase, New England Biolabs, #M0247S) for 10 min at 37C. Then, annealed, double-stranded T7 adapters (with unique sample barcodes) were ligated to chromatin DNA fragments (2 hours at 25C). Samples were pooled and split for incubation with the following histone modification antibodies at 4C overnight - Histone H3 (H3) antibody (#39763, Active Motif), Histone H3 lysine 4 monomethylation (H3K4me1) antibody (#ab8895, Abcam), Histone H3 lysine 4 trimethylation (H3K4me3) antibody (#ab8580, Abcam), Histone H3 lysine 9 trimethylation (H3K9me3) antibody (#ab8898, Abcam), Histone H3 lysine 27 acetylation (H3K27ac) antibody (#39133, Active Motif), Histone H3 lysine 27 trimethylation (H3K27me3) antibody (#07-449, Millipore) and Histone H3 lysine 36 trimethylation (H3K36me3) antibody (#61101, Active Motif). Protein G Dynabeads (Thermo Fisher, #10009D) were used to isolate and purify the chromatin fragments bound to each antibody, and further digested with proteinase K (Invitrogen, #25-530-049) for 1 hour at 63C. DNA clean-up and purification was done using 1X SPRIselect bead based reagent (Beckman Coulter, # B23318 retains fragments >100 bp). In vitro transcription was performed using HiScribe T7 Quick High Yield RNA Synthesis kit (New England BioLabs, #E2050S). Transcribed RNA was purified with MyOne Silane Dynabeads (Thermo Fisher, #37002D) and then reverse transcribed using a custom SBS3-random hexamer primer (IDT, CBE100-SBS3-RT) with SuperScript III First-Strand Synthesis SuperMix (Thermo Fisher, #18080-400). cDNA was purified again using SPRIselect beads and libraries were prepared using Terra Direct PCR Polymerase Mix (TakaraBio, # 639271) with P7_bc primer (forward with antibody-specific barcode) and P5_SBS3 (reverse) primer. Double size selection of libraries was performed using SPRIselect beads (selects >150 bp and <800 bp fragments). The libraries were sequenced using NovaSeq 6000 (Illumina) with an S4 flow cell in 150-bp PE configuration.
identifiers.namespace: SUB15503266
identifiers.id: 546421cbf94adc40