GSM3356523: MEF_H3K4me1_PIRCh_Rep1; Mus musculus; OTHER
To harvest the cells for PIRCh-seq. Approximately 4x107 cells were trypsinized and pooled into 50ml falcon tube. After wash with 40ml of cold PBS once. Make fresh 1% glutaraldehyde in room temperature PBS from 25% stock, discard remaining stock. Resuspend cell pellet in 1ml of glutaraldehyde solution and use a p1000 pipette to resuspend well, and top up to 40ml (1ml 1% glutaraldehyde / 1 milion cells). After invert a couple times, shake gently for 10 min, and then quench with 1/10 volume of 1.25M glycine. Invert a couple times, shake gently for 5 min, spin down 2000g, 4 min. Wash pellet once with 40ml cold PBS. Resuspend pellet in 1ml/20million cells of cold PBS. Aliquot 1ml to each fresh eppendorf tube, spin down 2000g, 4 min. Last, aspirate carefully, flash freeze cell pellets, store at -80C if necessary.For sonication, prepared cell pellets were spined down 2000g 4 min and remove any remaining PBS. Lysis per 20 million cells with 1ml of lysis buffer (1% SDS, 50mM Tris 7.0, 10mM EDTA, 1mM PMSF, 0.1U/ul Superase-in (Ambion), 1x Proteinase inhibitor (Roche)). After that, sonicate till chromatin size is ~300-2000bp and lysate is clear. Then spin down lysates at 16000g for 10 min. After that, we can flash freeze the supernate and store in -80℃ if necessary. For PIRCh-seq library construction, Add 400ul dilution buffer to each reaction and H3 or specific Histone modification antibody (Dilution buffer: 0.01% SDS, 1.1% Triton X 100, 1.2 mM EDTA, 16.7 mM Tris 7.0, 167 mM NaCl, 1mM PMSF, 0.1U/ul Superase-in (Ambion), 1x Proteinase inhibitor (Roche)). Shake the reaction end-to-end at 4℃ overnight. Use 50ul Protein A dynabeads per 5ug antibody IP. Wash beads with 5 times original volume of dilution buffer 4 times. Notice that do not exceed 200ul original volume of beads per tube. At last wash, aliquot beads to 1 tube per reaction. Aspirate buffer, resuspend and transfer beads to your IP using 200ul of the IP sample. End to end shake at room temperature for 2 hours. Wash with 1ml wash buffer 4 times, resuspend beads in 50ul IP elution buffer, vortex at setting 1 for 15 min. Then transfer supernatant to a fresh tube. Repeat elution on beads for once more. Combine supernatant for a total of 100ul. Immediatley add 5ul 3M NaOAc to neutralize pH. Add 10ul TurboDnase buffer and 1ul TurboDnase (Ambion), 37C for 30min. Add 3ul 500mM EDTA to eliminate divalent ions. Add 5ul Proteinase K (Ambion), 50C 45 min. For making sequencing libraryies, extract RNA using Trizol/chloroform, and precipitate with equal volume of isopropanol. Wash RNA pellet in 1ml ul 70% EtOH, resuspend pellets in 10ul H2O. Add 1ul TurboDnase buffer, Add 1ul TurboDnase, 1.2ul TurboDnase inactivating reagents. Vortex for 3', spin down. Save the 10ul supernatant, heat at 75C for 10' to kill Dnase. Use 5ul in Nugen Ovation v2 kit.