Nebnext index primers

Total RNA from WT, PB2 KO, and PB2 OE hESCs were isolated using an RNeasy Micro kit (Qiagen), following the recommended instructions for on-column DNase digestion. RNA quality and concentrations were measured using an Agilent Bioanalyzer (Agilent RNA 6000 Pico), and samples with RNA integrity number scores higher than 7 were used for the experiments. Poly(A) mRNA were isolated using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB E7490S) and cDNA libraries for Illumina next-generation sequencing were prepared with NEBNext Ultra Directional RNA Library Prep Kit (NEB E7760S). Three independent samples for each condition were barcoded using NEBNext Index primers (NEB, E7335S), pooled together as one sample, and ran on an Illumina platform HiSeq2500 with one-sided 75 base reads, with about 30 million reads per sample.

EBNA2 was immunoprecipitated from 30 × 10⁶ cross-linked GM12878 cells as described previously using the PE2 mouse monoclonal antibody and a rabbit anti-mouse secondary antibody (15 (link),16 (link)). A control immunoprecipitation was carried out in parallel using a 1:1 mix of sheep and mouse IgG (Dako). Libraries were prepared using the NEBNext ChIP-seq library prep reagent set for Illumina and NEBNext Index primers (New England Biolabs) and samples subjected to 50 bp single-end read sequencing with an Illumina Genome Analyzer IIx with a total of seven samples per lane. Data analysis was performed as described previously (15 (link),16 (link)). Data are available via GEO accession number GSE76869.

Ultra-low-input native ChIP-seq libraries were prepared by following a published protocol (Brind’Amour et al., 2015 (link)) using sorted wildtype (3268 cells) and Setdb1 cKO (5203 cells) early-to-mid pachytene cells. Briefly, sorted cells were treated with micrococcal nuclease at 21°C for 7.5 minutes and used for H3K9me3 ChIP. 5% of the samples after micrococcal nuclease digestion were taken as input. 6.25 μl of 1:100 diluted H3K9me3 antibody (Active Motif, 39161) per reaction was used for ChIP. DNA from pulled-down chromatin was eluted, end-repaired, phosphorylated, and A-tailed. NEBNext Adapter for Illumina was used as adapters and adapter-ligated DNAs were amplified by 13 PCR cycles using NEBNext Index primers (New England Biolabs, included in E7335 and E7500). The libraries were size-selected (200-500 bp) using E-Gel (ThermoFisher). Sequencing was performed on the Illumina HiSeq 2500 system (single read, 75 bp).