Agencourt ampure xp bead clean up

4C templates were prepared as previously described (24 , 62 (link)) with slight modifications. The outline of the 4C-seq procedure, viewpoint selection, and primer position are shown in Fig. S1, A and B. In brief, ∼1 × 10⁷ million cells were harvested and crosslinked with 2% formaldehyde for 10 min at RT. The nuclei were digested using primary enzyme Dpn II (RE1) (New England Biolabs) overnight. Following proximity ligation, DNA was reverse crosslinked and purified using phenol/chloroform extraction, and the ligated circular DNA was precipitated with ethanol. The ligated circular DNA was digested with secondary enzyme Csp6 I (RE2) (New England Biolabs) overnight, followed by proximity ligation and purification to obtain the 4C library. The 4C-seq library was generated by performing a two-step PCR using Phusion DNA polymerase (Thermo Scientific; # F530S). The primers of the 4C-seq library are listed in Table S2. For each 4C-seq library, we perform 32-tube PCR reactions, and each PCR reaction uses 100 ng DNA as a template. PCR reactions were pooled and purified with the QIAquick PCR purification kit (Qiagen; #28104). DNA smears from 200 bp to 1000 bp were extracted by agarose gel and subjected to Agencourt AMPure XP Bead clean-up (Beckman Coulter; #A63881) using a 0.8 bead/DNA sample ratio. The 4C-seq libraries were sequenced on the Illumina NovaSeq 6000 platform.

RNA was isolated using MyOne Silane Dynabeads (Thermo Fisher Scientific) and then fragmented and barcoded using eight-base pair barcodes together with standard Illumina adaptors. The primers were removed using Agencourt AMPure XP bead cleanup (Beckman Coulter/Agencourt), and the samples were amplified with 14 PCR cycles. Libraries were gel purified and quantified using a Qubit high-sensitivity DNA kit (Invitrogen), and library quality was assessed using TapeStation high-sensitivity DNA tapes (Agilent Technologies). RNA-seq reactions were sequenced on an Illumina NextSeq sequencer (Illumina) according to the manufacturer’s instructions, sequencing 50-base pair reads. Analysis was performed using the CLC Genomics Workbench v.8.0.1 RNA-seq analysis software package (Qiagen). Briefly, reads were aligned (mismatch cost = 2, insertion cost = 3, deletion cost = 3, length fraction = 0.8, similarity fraction = 0.8) to the mouse genome, and differential expression analysis was performed (total count filter cutoff = 5.0). The results were normalized to reads per million. Gene-e (Broad Institute) was used to generate heat maps. Datasets generated during the current study are available on the Gene Expression Omnibus (GSE134153) and are also available from the corresponding author upon reasonable request.