title: GSM5175935: RNA-Seq of Mus musculus: En2-KO-RA-1d rep-1; Mus musculus; RNA-Seq
library_construction_protocol: 1. For RNA -seq: cells were lysed with Trizol reagent (Life Technologies) and RNA was extracted according to the manufacturer's instructions. 2. For Capture-C：Briefly, cells were fixed with 1% (vol/vol) formaldehyde for 10 min at room temperature, quenched with 125 mM glycine in PBS, and then lysed in cold lysis buffer (10 mM Tris–HCl, pH7.5, 10 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 0.2% NP-40, 1× complete protease inhibitor cocktail (Roche)). Chromatin was digested with DpnII (New England Biolabs) at 37°C overnight. Fragments were then diluted and ligated with T4 DNA ligase (Takara) at 16°C overnight. Cross-linking was reversed by overnight incubation at 60°C with proteinase K (Bioline). Then 3C libraries were purified by phenol-chloroform followed by chloroform extraction and ethanol-precipitated at −80°C overnight. Sequencing libraries were prepared from 10μg of the 3C library by sonication to an average size of 200 ~300 bp and indexed using NEBnext reagents (New England Biolabs), according to the manufacturer's protocol. Enrichment of 2μg of an indexed library incubated with 3uM of a pool of biotinylated oligonucleotides (probe sequences are listed in Supplementary Table 7) was performed using the SeqCap EZ Hybridization reagent kit (#05634261001, Roche/NimbleGen), following the manufacturer's instructions. Two rounds of capture employing 48~72 and 24 hr hybridizations, respectively, were used. Correct library size was confirmed by agarose gel electrophoresis, and DNA concentrations were determined using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific). All sequencing was performed on Hi-Seq 2500 platforms using paired 150 bp protocols (Illumina). The Capture-C libraray was constructed with NEBNext DNA Library Prep Master Mix Set for Illumina (NEB).