Instrument: Illumina NovaSeq 6000
Strategy: Bisulfite-Seq
Source: GENOMIC
Selection: RANDOM
Layout: PAIRED
Construction protocol: RNA was harvested using Rneasy mini plus kit (Qiagen). 1.0 ug of total RNA was used for the construction of sequencing libraries.Purified genomic DNA (30 ng, spiked in with 5% of unmethylated lambda genomic DNA, Promega) was sheared to 200–500 bp using Covaris M220 (Covaris, MA). RNA libraries for RNA-seq were prepared using NEBNext Ultra Directional RNA Library Prep Kit (NEB #7760) following manufacturer's protocols. WGBS: The shared genomic DNA were then performed subjected t using EZ DNA methylation-lightning kit (Zymo Research) according to manufacturer's instructions. The bisulfite treated genomic DNA were further proceeded to library preparation with xGenTM Methyl Seq DNA Library Prep Kit (IDT, CA) following the manufactures' instructions. DamID:1 µg genomic DNA from mESCs expressing DamID only, TET2CD-Dam or TET2CDΔLCI-Dam is extracted and digested with DpnI (NEB,R0176) for 12 hrs at 37 °C. The digested DNA was purified by QIAquick PCR Purification Kit (Qiagen) to exclude uncut genomic DNA. Adaptors are ligated to the DpnI cutting fragments for PCR amplification. After digested with DpnII (NEB,R0543), DNA was then used as template for a PCR reaction to amplify the methylated fragments. DNA was then purified and sonicated into 200-300 bp for end repair, 3' end adenylation and sequencing adaptor ligation. The enriched libraries are pooled and subjected to high-throughput sequencing. Micro-C:Micro-C libraries were prepared with Micro-C Kit (Dovetail, CA) following the manufacturer's instructions. In brief, 1 million cells were resuspended and fixed by freshly made DSG (3 mM) for 15 min and then formaldehyde (1%) for another 10 min at room temperature. The crosslinking reaction was quenched with Tris buffer (pH = 7.5) to the final 0.75 M at room temperature. Fixed cells were washed twice and then subjected to chromatin fragmentation via MNase digestion for 15 min. After end repairing and proximity ligation, DNA were purified with streptavidin beads and adaptor ligation was performed on beads. An optimal PCR cycle for final library amplification was determined by quantification PCR and libraries were pooled and sequenced on Illumina Nova-seq platform with paired-end, 150/8/8/150 mode.