GSM3041218: C57Bl6_Thiomac_GRO_KLA-1h_SD_16-08-16-SD273; Mus musculus; OTHER
Nascent transcription was captured by global nuclear run-on sequencing (GRO-seq). Nuclei isolation. Nuclei were isolated from TGEMs using hypotonic lysis (10 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 3 mM CaCl2; 0.1% IGEPAL CA-630) and flash frozen in GRO-freezing buffer (50 mM Tris-HCl (pH 7.8), 5 mM MgCl2, 40% Glycerol). Run-on. 3-5 x 106 BMDM nuclei were run-on with BrUTP-labelled NTPs as described (Duttke et al., 2015) with 3x NRO buffer (15mM Tris-Cl (pH 8.0), 7.5 mM MgCl2, 1.5 mM DTT, 450 mM KCl, 0.3 U/uL of SUPERase In, 1.5% Sarkosyl, 366 uM ATP, GTP (Roche), Br-UTP (Sigma 40 Aldrich) and 1.2 uM CTP (Roche, to limit run-on length to ~40 nt)). Reactions were stopped after five minutes by addition of 500 uL Trizol LS reagent (Invitrogen), vortexed for 5 minutes and RNA extracted and precipitated as described by the manufacturer. Fragmentation. RNA pellets were resuspended in 18 ul ddH2O + 0.05% Tween (dH2O+T) and 2 ul fragmentation mix (100 mM ZnCl2, 10 mM Tris-HCl (pH 7.5)), then incubated at 70°C for 15 minutes. Fragmentation was stopped by addition of 2.5 ul 100 mM EDTA. BrdU enrichment. BrdU enrichment was performed using BrdU Antibody (IIB5) AC beads (Santa Cruz, sc-32323 AC, lot #A0215 and #C1716), as described in detail by Hetzel et al (Hetzel et al., 2016). Beads were washed once with GRO binding buffer (0.25×saline-sodium-phosphate-EDTA buffer (SSPE), 0.05% (vol/vol) Tween, 37.5 mM NaCl, 1 mM EDTA) + 300 mM NaCl followed by three washes in GRO binding buffer and resuspend as 25% (vol/vol) slurry with 0.1 U/uL SUPERase-in. To fragmented RNA, 500 uL cold GRO binding buffer and 40 uL equilibrated BrdU antibody beads were added and samples slowly rotated at 4°C for 80 minutes. Beads were subsequently spun down at 1000xG for 15 seconds, supernatant removed and the beads transferred to a Millipore Ultrafree MC column (UFC30HVNB; Millipore) in 2x 200 uL GRO binding buffer. The IP reaction was washed twice with 400 uL GRO binding buffer before RNA was eluted by incubation in 200 uL Trizol LS (Thermo Fisher) under gentle agitation for 3 minutes. The elution was repeated a second time, 120 uL of dH2O+T added to increase the supernatant and extracted as described by the manufacturer. End repair and decapping. For end-repair and decapping, RNA pellets were dissolved in 8 ul TET (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.05 % Tween20) by vigorous vortexing, heated to 70°C for 2 minutes and placed on ice. After a quick spin, 22 ul Repair MM (3 ul 10x PNK buffer, 15.5 ul dH2O+T, 0.5 ul SUPERase-In RNase Inhibitor (10 U), 2 ul PNK (20U), 1 ul RppH (5U)) was added, mixed and incubated at 37°C for 1 hour. 5' Phosphorylation. To phosphorylate the 5'end, 0.5 ul 100 mM ATP was subsequently added and the reactions were incubated for another 45 minutes at 37°C (the high ATP concentration quenches RppH activity). Following end repair, 2.5 ul 50 mM EDTA was added, reactions mixed and then heated to 70°C for 2 minutes before being placed on ice. A second BrdU enrichment was performed as detailed above. Adapter Ligation. RNA pellets were dissolved in 2.75  TET + 0.25  Illumina TruSeq 3'Adapter (10  M), heated to 70°C for 2 minutes and placed on ice. 7  of 3'MM (4.75  50% PEG8000, 1  10x T4 RNA ligase buffer, 0.25  SUPERase-In, 1  T4 RNA Ligase 2 truncated (200U; NEB)) was added, mixed well and reactions incubated at 20°C for 1 hour. Reactions were diluted by addition of 10  TET + 2 ul 50 mM EDTA, heated to 70°C for 2 minutes, placed on ice and a third round of BrdUTP enrichment was performed. RNA pellets were transferred to PCR strips during the 75% ethanol wash and dried. Samples were dissolved in 4  TET (10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 0.05% Tween 20) + 1  10  M reverse transcription (RT) primer. To anneal the RTprimer, the mixture was incubated at 75°C for 5 minutes, 37°C for 15 minutes and 25°C for 10 minutes. To ligate the 5' Illumina TruSeq adapter, 10  5'MM (1.5  ddH2O + 0.2% Tween20, 0.25  denaturated 5'TruSeq adapter (10  M), 1.5  10x T4 RNA ligase buffer, 0.25  SUPERase-In, 0.2  10 mM ATP, 5.8  50% PEG8000, 0.5  T4 RNA ligase 1 (5U; NEB)) was added and reactions were incubated at 25°C for 1 hour. Reverse Transcription. Reverse transcription was performed using Protoscript II (NEB) (4  5x NEB FirstStrand buffer (NEB; E7421AA), 0.25  SUPERase-In, 0.75  Protoscript II (150U; NEB)) at 50°C for 1 hour. After addition of 30  PCR MM (25  2X LongAmp Taq 2X Master Mix (NEB), 0.2  100  M forward primer, 2.8  5M Betaine and 2  10  M individual barcoding primer), mixtures were amplified (95°C for 3 minutes, (95°C for 60 seconds, 62°C for 30 seconds, 72°C for 15 seconds) x13, 72°C for 3 minutes). PCR reactions were cleaned up using 1.5 volumes of SpeedBeads™ (GE Healthcare) in 2.5M NaCl/20% PEG8000 Library selection and cleaning. Libraries were size selected on a PAGE/TBE gels to 160-225 base pairs. Gel slices were shredded by spinning through a 0.5 ml perforated PCR tube placed on top of a 1.5 ml tube. 150 ul Gel EB (0.1% LDS, 1M LiCl, 10 mM Tris-HCl (pH 7.8)) was added and the slurry incubate under agitation overnight. To purify the eluted DNA, 700 ul Zymogen ChIP DNA binding buffer was added into the 1.5 ml tube containing the shredded gel slice and the Gel EB, mixed by pipetting and the slurry transferred to a ZymoMiniElute column. Samples were first spun at 1000xG for 3 minutes, then 10,000xG for 30 seconds. Flow through was removed, and samples washed with 200 ul Zymo WashBuffer (with EtOH). Gel remainders were removed by flicking and columns washed by addition of another 200 ul Zymo WashBuffer (with EtOH). Flow through was 42 removed, columns spun dry by centrifugation at 14,000xG for 1 minute and DNA eluted by addition of 20 ul pre-warmed Sequencing TET (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 0.05% Tween 20). Libraries were sequenced. RNA pellets were resuspended in 18 ul ddH2O + 0.05% Tween (dH2O+T) and 2 ul fragmentation mix [100 mM ZnCl2, 10 mM Tris-HCl (pH 7.5)], then incubated at 70°C for 15 minutes. Fragmentation was stopped by addition of 2.5 ul 100 mM EDTA. BrdU enrichment was performed using BrdU Antibody (IIB5) AC beads (Santa Cruz, sc-32323 AC, lot #A0215 and #C1716), as described in detail by Hetzel et al (Hetzel et al., 2016). Beads were washed once with GRO binding buffer [0.25×saline-sodium-phosphate-EDTA buffer (SSPE), 0.05% (vol/vol) Tween, 37.5 mM NaCl, 1 mM EDTA] + 300 mM NaCl followed by three washes in GRO binding buffer and resuspend as 25% (vol/vol) slurry with 0.1 U/uL SUPERase-in. To fragmented RNA, 500 uL cold GRO binding buffer and 40 uL equilibrated BrdU antibody beads were added and samples slowly rotated at 4°C for 80 minutes. Beads were subsequently spun down at 1000xG for 15 seconds, supernatant removed and the beads transferred to a Millipore Ultrafree MC column (UFC30HVNB; Millipore) in about 2x 200 uL GRO binding buffer. The IP reaction waswashed twice with 400 uL GRO binding buffer before RNA was eluted by incubation in 200 uL Trizol LS (Thermo Fisher) under gentle agitation for 3 minutes. The elution was repeated a second time, 120 uL of dH2O+T added to increase the supernatant and extracted as described by the manufacturer. For end-repair and decapping, RNA pellets were dissolved in 8 ul TET [10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.05 % Tween20] by vigorous vortexing, heated to 70°C for 2 minutes and placed on ice. After a quick spin, 22 ul Repair MM [3 ul 10x PNK buffer, 15.5 ul dH2O+T, 0.5 ul SUPERase-In RNase Inhibitor (10 U), 2 ul PNK (20U), 1 ul RppH (5U)] was added, mixed by flicking and incubated at 37°C for 1 hour. To phosphorylate the 5'end, 0.5 ul 100 mM ATP was subsequently added and the reactions were incubated for another 45 minutes at 37°C (the high ATP concentration quenches RppH activity). Following end repair, 2.5 ul 50 mM EDTA was added, reactions mixed and then heated to 70°C for 2 minutes before being placed on ice. A second BrdU enrichment was performed as detailed above. For li