title: GSM4586183: WT_EloA_cutnrun_rep2; Mus musculus; OTHER
library_construction_protocol: Bru-RNA enrichment:Mouse aBrdU antibody (SCBT, sc-32323) was coupled to Dynabeads M-280 Sheep Anti-Mouse IgG (11201D). 1ml of magnetic beads was washed 4 x with PBS-DEPC/0.1%BSA and 500ul of Ab  was incubated with 1ml of beads and yeast RNA (500ng/µl final concentration) for 2-3 hours at 4oC, washed 3x with PBS-DEPC/0.1%BSA/Tween 0.05%.The final volume was raised to 1ml. For enrichment of Bru-RNA, 25-50µg of total RNA (after preheating at 95oC and immediate cooling) was added to 50µl of Antibody-bound beads in 1 ml of PBS-DEPC/0.1%BSA/Tween 0.05% and incubated for 90 min at RT with a foil wrapped around the tube. Beads were washed 4x with PBS-DEPC/0.1%BSA/Tween 0.05%, 5-10min (last wash was done with Tween-free buffer). Enriched RNA was either eluted by addition of 25µl of DEPC-water and incubation at 95oC for 10min with occasional flicking/shaking, or Trizol extraction. The enriched RNA was depleted of ribosomal RNA and used for generation of RNA-seq library. ChIP-seq: mESC were MEF depleted, crosslinked with 1% formaldehyde in PBS for 10min in conical tubes while gently rotating at RT and quenched by adding Glycine to a final concentration of 0.125M and left at RT for 5min on rotator. Crosslinked cells were spun down for 3min at 3500 rpm (2850 rcf) and lysed in cell lysis buffer  (50mM Tris PH 8.0, 50mM NaCl, 1mM EDTA, 5mM MgCl2, 0.25% NP-40, Roche complete EDTA-free protease inhibitor tablet(PI)* 0.2% PMSF* and 0.2% DTT*(*freshly-added)) at 2-2.5x10e7 cells/ml. Cells were left on ice for 10 min and dounced 10x using the tight pestle. Nuclei was spun down at 2850 rcf in 15ml conical tubes and the supernatnant was completely removed and nuclei was resuspended in sonication buffer (20mM Tris PH 8.0, 1% SDS, 2mM EDTA, 0.5mM EGTA, 0.5mM PMSF*, PI*(freshly added) at 5.0x10e7 nuclei/ml. Resuspended nuclei was left on ice for 10min and sonicated using the Bioruptor sonicator, at high setting 30'' on 45'' off twice for 15min. Water and ice was changed in between each sonication. IP was performed with 10 ul of  EloA R-19 antibody (SCBT, sc-1557), 1-2.5x10e6 nuclei were used per IP reaction, by adding 20-50 µl of sonicated chromatin to 1-1.25ml of IP buffer (20mM Tris PH 8.0, 0.5% Triton X-100, 150mM NaCl, 10% glycerol, 2mM EDTA) with the respective antibodies and after 4 hours of incubation protein A (40-50 µl) was added to the IP reaction and incubated for 4 more hours. Each wash was done on the rotator at RT for 5min as follows: x1 low salt wash (20mM Tris PH 7.8, 0.1% SDS, 1% Triton X-100, 2mM EDTA, 150mM NaCl), x3 high salt wash (20mM Tris PH 7.8, 0.1% SDS, 1% Triton X-100, 2mM EDTA, 500mM NaCl), x1 LiCl wash (20mM Tris PH 7.8, 1% NP-40, 1% NaDeoxycholate, 2mM EDTA, 250mM LiCl), x2 TE wash.Elution was done on the IPed samples in 2x250µl of elution buffer (1% SDS, 0.1M NaHCO3) on the rotator at RT. To the 500µl elution buffer, 20 µl of 5M NaCl was added and samples were incubated in a thrmomixer for 4hr-O/N at 65oC to reverse the crosslink (same was done for the Input material). Next, 15µl of 1M Tris PH 7.8, 1-2 µl glycoblue (Ambion) and 2 µl of proteinase K was added to the reaction mix and incubated at 65oC for 30-60min. DNA was extracted from the reversed crosslink material using phenol/chloroform extraction and EtOH precipitation. Cut-and-run-seq:Mouse ES cells were crosslinked with 1% formaldehyde in PBS for 10 min and quenched with 0.125M glycine for 5min, cells were pelleted and washed once with PBS, spun down, resuspended in freezing media (DMEM, 10% DMSO), placed in a styrofoam container and frozen O/N (this step was included to minimize cell membrane rupture and DNA breakage). On the day of the experiment, crosslinked cells were thawed and washed once with PBS and twice in cut&run wash buffer (20 mM HEPES pH7.5, 150 mM NaCl, 0.1% Tween20, 0.1%BSA, 1 Roche complete EDTA-free protease inhibitor tablet/50ml), Tween20 was included in the wash buffer to minimize  concanavalin A magnetic bead aggregation in downstream steps. Next, 5x10e6 cells were resuspended in 950 μl of wash buffer. 50 μl of BioMag-Plus Concanavalin A beads (Polysciences Inc.) was also washed gently twice with binding buffer (20 mM HEPES pH 7.9, 1 mM CaCl2, 1mM MnCl2) and resuspended in binding buffer at the initial volume (50 μl). Concanavalin A beads (50μl) was added to 5x10e6 cells in 950 μl of wash buffer and incubated rotating for 15min at RT. The cells were divided into five 200ul aliquots for each antibody used (1x10e6 cells/antibody) and after removal of wash buffer, 50 μL of ice cold antibody buffer (wash buffer containing 0.05% Digitonin and 2mM EDTA) containing 0.5 μg of antibody was added to 1x10e6 bead-bound cell pellet and the mixture was gently resuspended by pipetting up and down 25 times . Resuspended beads were placed on a vortex at the lowest setting, and incubated at room temperature for 15 min. For the EloA cut&run experiment using goat anti EloA (SCBT, R-19), a bridging rabbit anti goat antibody was used for efficient binding to protein A. Cells were washed once after primary antibody incubation with 1ml of Digitonin buffer (Wash buffer containing 0.05% Digitonin) resuspended in 50 μl of digitonin buffer with 0.5 μg of rabbit anti goat IgG (rabbit anti-goat IgG (ab6697)). Left on the lowest setting of the vortex and incubated at RT for 15 min. Next, permeabilized, antibody-bound cells were washed once in digitonin buffer containing 1mM EDTA, and 50 μL of digitonin buffer containing Protein A-MNase (pA-MN) at a final concentration of 700ng/ml was added to the pellet, resuspended and incubated at room temperature for 15min. Cells were washed twice with digitonin buffer containing 1mM EDTA. Next, 150 μl of ice cold digitonin buffer was added to the pA-MN bound cells, resuspended and digested on ice (0 oC) for 30 min after addition of CaCl2 to a final concentration of 4 mM. Added 100ul of 2x stop buffer as described in (Skene et al, 2018), and incubated on thermomixer for 15min at 37oC to release the cleaved DNA fragments into solution. The mixture was spun down in a cold tabletop centrifuge at max speed for 5min, and 2ul of proteinase K was added to the supernatant eluate. The mixture was incubated at 65 oC for 4 hours and DNA was extracted by using ChIP DNA Clean & Concentrator (Zymo research). Library constructing for sequencing was prepared as described elsewhere (Bowman et al., BMC Genomics 2013).