T4 dna ligase

Bar-coded libraries were created as described by Andolfatto et al. [55 (link)] with several modifications, with 6-bp barcoded adapters designed and modified according to the standard Illumina adapter protocol for paired-end read libraries. DNA was enzyme digested using FastDigest Taqα^I (Thermo scientific Fermentas) and adapter ligation was carried out with T4 DNA ligase (Enzymatics). Subsequently, 19 to 24 of the resulting samples with different indexes were pooled together. DNA fragments between 400 and 600 bp were retrieved and purified using a QIAquick Gel Extraction kit (Qiagen, Valencia, CA). After purification, the adapter-ligated DNA fractions were subjected to PCR amplification (Phusion High-fidelity, Finnzymes). The PCR products were gel separated, and the 400–600-bp DNA fractions were purified using a QIAquick PCR Purification kit (Qiagen, Valencia, CA). Finally, the purified libraries were quantified on an Agilent 2100 Bioanalyzer and sequenced on an Illumina HiSeq 2000 instrument at BGI, Shenzhen, China.

Genomic DNA (200 ng) were bisulfite converted using EZ-DNA Methylation-Gold Kit (Zymo, #D5006) according to the manufacturer’s protocol. After conversion, the DNA were subjected to a single-stranded library preparation protocol Tequila 7N (Euler Technology). In brief, the DNA were end-repaired using Klenow (NEB) and tailed with poly-A using TdT (Takara), ligated to a poly-T overhang adaptor using T4 DNA ligase (Enzymatics), and linearly amplified for 12 cycles using PhusionU (Thermo Fisher Ltd.). The linear products were then annealed to a 5' adaptor with 7 bp 3' random nucleotide overhang and amplified using adaptor oligos (Sangon, Shanghai, China) with Phusion (Thermo Fisher Ltd.), resulting in a library with proper Illumina sequencing adaptor ends ready for NGS. Hybridization was done with SeqCap EpiGiant Enrichment Probe (Roche, #07138911001), oligos and SeqCap wash and binding buffers (Roche) following the manufacturer’s protocol. After hybridization, the library was amplified using Phusion (Thermo Fisher Ltd.) for 8 cycles and sequenced on Novaseq6000 sequencer (Illumina, CA) to 100 M PE150 reads. Sequencing was performed at Berry Genomics, Beijing, China. QC metrics is described in Additional file 9: Table S8.

CUT&RUN was performed as previously described (30 (link),31 (link)) using EZH2, H3K27me3 and IgG antibodies on 5 million cells per sample. DNA obtained from Drosophila melanogaster S2 cells was sonicated to 200bp and 1ng was added to each CUT&RUN sample as a heterologous spike-in control. ChIP was performed as previously described (32 (link)) using 9 μg of ATRX antibody (21 (link)) and 6 million cells per sample. For RNA-sequencing, RNA samples were extracted from Day 0 mESCs and Day 6 NPCs using Trizol reagent (Invitrogen) and subjected to DNase digestion with Turbo DNase (Ambion AM2238), then rRNA-depleted using FastSelect -rRNA HMR (Qiagen) and converted to cDNA using Ultra II Directional RNA Library Prep Kit (NEB E7760). DNA and cDNA samples were end-repaired using End-Repair Mix (Enzymatics), A-tailed using Klenow exonuclease minus (Enzymatics), purified with MinElute columns (Qiagen), and ligated to Illumina adapters (NEB #E7600) with T4 DNA ligase (Enzymatics). Size selection for fragments >150 bp was performed with AMpure XP (Beckman Coulter). Libraries were PCR amplified with barcoded adapters for Illumina sequencing (NEB #E7600) using Q5 DNA polymerase (NEB #M0491) and purified with MinElute. Sequencing was performed on a NextSeq 500 instrument (Illumina) with 38 × 2 paired-end cycles.

Total RNA was isolated with hot acidic phenol as described above. Library preparation was performed as described^{53 (link)} with the addition of an ERCC spike-in mix for two biological replicates. Briefly, total RNA was fragmented and reverse transcribed with random hexamers. Following dUTP incorporation, cDNAs were end-repaired with a mix of end-repair enzymes (T4 DNA Polymerase (3 U/μl, NEB), Klenow DNA Polymerase (5 U/μl, NEB), and T4 PNK (10 U/μl, NEB)) and A-tailing was performed. Adapter ligation was done using T4 DNA ligase (600 U/μl, Enzymatics, Inc.). After UDG treatment, final PCR was performed with indexing primers. Size of the library was determined by Fragment Analyzer and the concentrations were determined by Qubit 4.0 fluorometer (Invitrogen). Sequencing of all samples was carried out on an Illumina NextSeq 500 with a read length of 150 (paired-end).