Universal dna library prep kit

Biotin ChIP-seq was performed as described (54 (link)). In brief, mESCs stably expressed Biotin–RBBP4 or Biotin–RBBP7 were cross-linked with 1% formaldehyde. The cross-linked cells were resuspended in SDS lysis buffer [1% SDS, 50 mM Tris–HCl (pH 8.0), 10 mM EDTA], sonicated and diluted 10-fold with ChIP dilution buffer [0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl (pH 8.0), 167 mM NaCl], and then incubated with M280 Streptavidin Dynabeads (Invitrogen, 11205D) at 4°C overnight. Streptavidin Dynabeads-bound DNAs were subsequently washed once with wash buffer 1 (2% SDS), once with wash buffer 2 [50 mM HEPES (pH 7.5), 1 mM EDTA, 500 mM NaCl, 0.1% sodium deoxycholate, 1% Triton X-100], once with wash buffer 3 [10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate] and then twice with TE wash buffer [10 mM Tris–HCl (pH 8.0), 1 mM EDTA]. ChIPed DNAs were reverse-cross-linked and purified. The sequencing libraries were prepared through the VAHTS Universal DNA Library Prep Kit (Vazyme, ND607) and sequenced on a NovaSeq 6000 sequencer.

Detail methods of DNA sequencing was performed as our previous study [12 (link)]. DNA panel was 2,189 kb in length, which covered full coding regions or hotspot mutation regions in 769 genes. The genomic DNA from formalin-fixed paraffin-embedded (FFPE) tumor samples was extracted using the MagPure FFPE DNA Kit B (Magen, China), fragmented into DNA pieces of approximately 200 bp using an enzymatic method (5 × FEA Enzyme Mix; Qiagen, CN), subsequently constructed DNA libraries using a VAHTS Universal DNA Library Prep Kit (Vazyme, China). The libraries of genomic DNA were captured with a 769 gene panel (Table S1) and sequenced using a HyperCap Target Enrichment Kit (Roche, Switzerland) and the NovaSeq 6000 system (Illumina, USA) according to the manufacturer’s protocols, respectively.