GSM1530533: WT_shTdg_Vc_E14_WG.MABseq; Mus musculus; Bisulfite-Seq
For whole genome (WG) MAB-seq analysis, genomic DNA was extracted from mESCs using the DNeasy Blood & Tissue Kit (Qiagen 69504). To capture H3K4me1- or H3K27me3-enriched chromatin for genome-scale MAB-seq/BS-seq analysis, ~15 million cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature followed by exposure to 0.125 M glycine. After two washes with cold PBS, cells were collected and stored at -80 °C before use. Chromatin were extracted and lysed sequentially with lysis buffer (LB3, 10 mM Tris-HCl, pH8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine). Chromatin was sonicated using a microtip (Branson sonifier 450) until the DNA fragments were reduced to 200-1000 bp in length. 10 μg antibodies were immobilized with 100 μl Dynal protein-G beads (Invitrogen) for at least 6 hours. Immunoprecipitation was performed overnight at 4 °C with antibody-conjugated protein-G beads. DNA/protein complexes were washed with RIPA buffer (50 mM Hepes-KOH, pH7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate) for five times and reverse cross-linked at 65 °C overnight. The DNA was treated sequentially with RNase A and proteinase K, and purified by phenol/chloroform extraction and ethanol precipitation. Following antibodies were used in ChIP assays: anti-H3K4me1 (ab8895, Abcam) and anti-H3K27me3 (07-449, Millipore). For genome-scale MAB-seq or BS-seq, 200-500 ng of ChIP DNA (H3K4me1- or H3K27me3-enriched) was first spiked-in with unmethylated lambda DNA (1:400), which was then repaired and ligated to methylated (5mC) custom adapters (forward 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3’; reverse 5’-/5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTC-3’; the asterisk denotes phosphorothioate bond) with the NEBNext Ultra DNA Library Prep kit from Illumina (NEB). Adaptor-ligated DNA was then purified with 1.2x AMPure XP beads (Beckman Coulter). For BS-seq, methylated-adpator-ligated DNA was directly subjected to bisulfite conversion using Qiagen EpiTect DNA Bisulfite Kit (Qiagen, 59104) per manufacturer’s instructions, except that the thermal cycle was repeated twice. For MAB-seq, methylated adpator-ligated DNA was treated by M.SssI (New England Biolabs, M0226M) in a 50-µL reaction for two rounds. In each round of treatment, DNA was first incubated with 1.0 unit/µL M.SssI methylase (New England Biolabs, M0226M) for 4 hours in 25-µL reaction [1.25 µL of 20 unit/µL M.SssI and 0.5 µL of 32 mM SAM (final concentration: 640 µM)], and additional 25-µL containing same concentration of M.SssI (1.0 unit/µL) and SAM (640 µM) was supplemented to treat DNA for another 8 hours (in 50-µL). Of note, the first round of M.SssI treatment was performed in Mg++-free reaction buffer [10 mM Tris-HCl (pH 8.0), 50 mM NaCl, 10 mM EDTA], while the second round was carried out with NEB buffer #2 [10 mM Tris-HCl (pH 7.9), 50 mM NaCl, 10 mM MgCl2, 1 mM DTT]. DNA was purified by sequential Phenol/Chloroform/Isoamyl Alcohol (PCI, 25:24:1) extraction and ethanol precipitation after each round of M.SssI treatment. M.SssI-treated, methylated adaptor-ligated DNA was then subjected to bisulfite conversion using the EpiTect DNA Bisulfite Kit (Qiagen) as described above. Bisulfite-treated DNA was pre-amplified for 5 cycles using KAPA HiFi Uracil+ HotStart ReadyMix (KAPA) with indexed and universal primers from NEBNext Multiplex Oligos for Illumina (Index Primers Set 1). The optimal PCR cycle numbers required to generate the final libraries were then determined by quantitative PCR. Final libraries were generated by scaled-up PCR reactions using the cycles determined above, and purified with 1.2x AMPure XP beads. For whole-genome MAB-seq analysis, 1 µg non-enriched genomic DNA was first spiked-in with unmethylated lambda DNA (1:400) and was then treated with M.SssI (1st round protocol). M.SssI-treated genomic DNA (in 50-µL) was fragmented to an average size of 300-400 bp with Covaris M220 (20% duty factor, 200 cycles per burst, 80 sec x 2) using microTUBE AFA Fiber Screw-Cap. Sheared DNA was purified (1.2x AMPure XP beads), end-repaired and ligated to methylated adapters as described for H3K4me1/H3K27me3-MAB-seq. Methylated-adaptor-ligated DNA was treated with M.SssI again (2nd round protocol) before bisulfite conversion (Qiagen EpiTect DNA Bisulfite Kit). Bisulfite-treated DNA was prepared for sequensing as described above.
GEO Accession
GSM1530533