title: GSM5700648: MS_hichip_rep1; Sus scrofa; OTHER
library_construction_protocol: Skeletal muscle are collected from 2-week-old LW and MS piglets. For BL Hi-ChIP, Approximately 0.2g of frozen muscle tissue was ground into a fine powder in liquid nitrogen and fixed with cross-linking buffer (1% formaldehyde, 0.1M NaCl, 1mM EDTA, 0.5mM EGTA and 50mM Hepes) for 15 min at room temperature. The cross-linking reaction is terminated by the addition of a final concentration of 0.2M glycine. Centrifugate at 2500rpm for 10min at 4°C to collect the fixed tissue and resuspended in tissue lysis buffer(10mM Tris-HCl pH8.0、10Mm NaCl、0.2% v/v Igepal CA630、1× protease inhibitors), incubated on ice for 30min to lyse the tissue and extract the nuclei. Filtering out impurities with a 40μl cell strainer to obtain nuclei of higher purity. The nuclei were resuspended in 1ML of SDS buffer (1×CutSmart and 0.5% SDS) and incubated for 10 min at 62°C, followed add 200µl 10% TritionX-100 and incubated at room temperature for 10 min to neutralise the SDS. Pelleting and washing with 1X Cutsmart buffer, chromatin was digested with 1U/μl AluI restriction enzyme at 37℃ for 7h. Collecting the nuclei and resuspending the nuclei in 500µl A -tailing solution and incubate for 1 hour at 37°C. Pellet again, resuspending the nuclei in 500µl ligation solution with biotinylated bridge linker and incubate over night at 16°C. Nuclear were fragmented by Covaris S220, 7.5µg antibody (H3K27ac, Abcam Cat# ab4729) and 30µl M-280 sheep anti-rabbit IgG Dynabeads (ThermoFisher, 11203D) were used to enrich the chromatin complexes mediated by H3K27ac. Purifying the ChiP-DNA and fragmenting with Tn5. Streptavidin C-1 beads were used to pull down the DNA fragement like “DNA-linker-DNA”.    GRID-seq was performed as described in Li et al. Approximately 2.5×106 cells were crosslinked for 45 min at room temperature with 2mM DSG solution and further fixed in 1% formaldehyde for 10 min. Fixed cells were permeabilized in 1ml equilibrium buffer (0.2% NP-40, 5mM Tris-HCl pH 7.5, 10 mM NaCl, and 1×protease inhibitor cocktail) for 15 min on ice with brief centrifugation). To prepare nuclei, fixed cells were incubated in 300 μL of Cutsmart buffer (0.5% SDS) for 10 min at 62°C. Then, 50ul of 10% Triton X-100 was added to quench the SDS, and it was chilled on ice. To digest chromatin, nuclei were collected by brief centrifugation, washed twice with 1× Cutsmat Buffer, resuspended in 500 μl of AluI solution (1× Cutsmart Buffer, 1% Triton X-100,1 U/μL RiboLock, 0.5 U/μL AluI (NEB), and 1× protease inhibitor), and incubated at 37 °C for 4 h. After digestion, DNA ends were added with dA tailing module, and then repaired with DNA/RNA 5'phosphorylation and 3'dephosphorylation using PNK (NEB) at 37°C for 1.5h.   To ligate in situ linkers to RNA, RNA ends were ligated using 4U/ul T4 RNA Ligase 2-truncated KQ (NEB) to an oligonucleotide 'bridge' molecule that contained a 5'-adenylated ssDNA overhang and incubated at 25 °C for 3 h. To ligate in situ linkers to DNA, nuclei were collected, washed twice with 800 μL of 1× DNA Ligase Buffer (NEB) to remove free linkers, resuspended in 1 ml of DNA Ligation Solution (0.2 U/μL RiboLock, 1× DNA Ligase Buffer, 1 mg/ml BSA, 1% Triton X-100, and 1 U/μL T4 DNA Ligase (NEB)) and incubated overnight at 16 °C. Next, the pellet was washed, and the RNA strand was stabilized by first strand synthesis of the RNA through extension of the bridge by Bst 3.0 polymerase.  Reversal of crosslinking and purification of DNA/RNA. The Pellet was resuspended in 300 ul proteinase K solution (15 mM EDTA, 10 mM Tris-HCl, pH 7.5, 0.5% SDS, and 1 mg/ml proteinase K (Ambion)), and incubate at 65˚C for at least 2 h in Thermomixer. After Adding 300μL of Phenol: Chloroform: Isoamyl Alcohol (pH 8.0) to each tube, Total DNA/RNA were precipitated with 2 μL of GlycoBlue, 30μL of 3 M NaOAc (pH 5.5), and 300μL of 100% isopropanol on ice for at least 2h, followed by centrifugation for 30min at 16000g. Total DNA/RNA were dissolved in 51 μL of H2O (expect a total amount of ~10ug DNA/RNA).   Pull down biotin DNA/RNA. 50ul DNA/RNA was added to M280 streptavidin magnetic beads which had been washed three with 1×B&W Buffer (5 mM Tris-Cl pH 7.5, 0.02% Tween-20, 0.5 mM EDTA, 1 M NaCl). After incubate for 45min at RT, the beads were washed with 500 ul of 1×B&W Buffer for 5 times, and resuspended in 100 μL freshly prepared 150mM NaOH at RT for 10min. Next, the supernatant was transferred to a 1.5 mL tube and neutralized with 11 μL 10 × TE buffer and 6.5 μL of 1.25M acetic acid. Single stranded DNA (ssDNA) was precipitated with 2μL of GlycoBlue,10 μL of 3 M NaOAc (pH 5.5), and 100 μL of 100% isopropanol. With Second strand synthesis, the reaction was performed by adding 0.5 μL of 10mM dNTP, and 5 U klenow Exo-(NEB) and incubated at 37°C for 1 h. Next, Synthetic double-stranded DNA were digested with MmeI buffer (5 pmol SAM(NEB), and 4 U MmeI) for 1h at 37°C. After MmeI digestion, DNA was extracted and purified, the 84bp target band was cut and used for Illumina TrueSeq library construction.