title: GSM6124020: RNA-Seq: EBd5-K5aSE-WT-Rep1; 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 4C-seq：Briefly, 10^7 ESCs were trypsinised into single cells and resuspended in 9.5 ml of DMEM/15% FBS. Cells were then cross-linked by adding 0.6 ml of 16% formaldehyde for 10 min. After centrifugation at 4 degrees (2000 g), cells were lysed in 5 ml of lysis buffer (10 mM Tris-HCl [pH 7.5]; 10 mM NaCl; 5 mM MgCl2; 0.1 mM EGTA; 1 x protease inhibitor) for 10 min (placed ice with constant light shaking) and centrifuged to remove the supernatant. The nuclei were then digested with DpnII at 37°C. After inactivation with 1.6% SDS for 20 minutes at 65°C, samples were diluted in 6.125 ml of 1.15× ligation buffer and 100 U T4 ligase and incubated for 4 hours at 16°C, followed by 30 minutes at 25°C. The ligated chromatin was digested by proteinase K, purified by phenol-chloroform extraction and the DNA was precipitated by ethanol. The purified product was further digested with NlaIII and then cyclised with T4 ligase. After purification, PCR reactions containing 100 ng DNA were performed using the bait-primers. PCR products were purified by Agencourt AMPure XP paramagnetic beads and the purified products were sequenced at Novogene (Tianjin city, China). 1. RNA-seq Library. Total RNA was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system. Then 3 µl USER Enzyme (NEB, USA) was used with size selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq 6000 platform and 150 bp paired-end reads were generated. 2. 4C-seq Library. After the DNA samples were tested and passed, the DNA fragments specifically enriched by the 4C technique were subjected to end repair, addition of A-tails, addition of sequencing connectors, purification and PCR amplification to complete the entire library preparation. After library construction, initial quantification was performed using Qubit 2.0 to dilute the library, followed by detection of the insert fragment using Agilent 2100. After the insert fragment size met the expectation, the effective concentration of the library was accurately quantified using Q-PCR method to ensure the quality of the library. After the libraries passed the assay, the different libraries were pooled into flowcell according to the effective concentration and the target downstream data volume. cBOT was clustered and sequenced using Illumina high-throughput sequencing platform NovaSeq 6000.