alias: GSM3595849
title: GSM3595849: PLAC-seq-NALM6-H3K4me3-2; Homo sapiens; OTHER
library_construction_protocol: PLAC-sequencing: 10M NALM6 cells that had been cross-linked with 1% formaldehyde for 10 minutes (see Chromatin immunoprecipitation section) were thawed on ice for 5 minutes, re-suspended in ice cold lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2% NP-40, 1X Roche protease inhibitors – 11697498001 (PIC)) and incubated on ice for 15 min. Samples were spun for 5 min @ 4C at 2,500g and cell pellet was washed in ice-cold lysis buffer + PIC. The pellet was resuspended in 0.5% SDS and incubated @ 62C for 10 min. The SDS reaction was quenched by addition of water and 10% Triton X-100 followed by 15min incubation @ 37C. The sample was digested with 40U MboI restriction enzyme with addition of 25 µl NEBuffer2 followed by 2h incubation @ 37C with shaking (900 RPM). After restriction enzyme digestion the samples were incubated for 20min @ 62C to inactivate MboI followed by a cool-down to RT. A fill in reaction was conducted using 0.3 mM Biotin-14-dATP (ThermoFisher, 19524016), 0.3 mM dCTP, 0.3 mM dTTP, 0.3 mM dGTP and 40U Klenow (NEB, M0210) @ 37C for 1.5h with shaking (900 RPM). Next a Ligation master mix (1X T4 ligation buffer (NEB, B0202), 1% Triton-X 100, 120 ug BSA (NEB, B9000), 4000 U T4 DNA ligase (NEB, M0202)) was added and samples were incubated @ RT with rotation for 2h. The samples were centrifugated 2,500g 5min @ 4C, supernatant was removed and cell pellet was resuspended in 250 µl ChIP SDS-lysis buffer (0.5% SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris-HCl (pH 8)) + PIC followed by 18 rounds of sonication (30s max intensity followed by 30s rest) with a Bioruptor (Diagenode). Sheared chromatin was centrifuged for 10 min @ 4C, 13000 rpm and supernatants were transferred to new tubes and diluted in 750 µl 1xHBSS + 1 ml 2X RIPA (20 mM Tris–HCl, pH 7.5, 2 mM EDTA, 2% Triton X-100, 0.1% SDS, 0.2% sodium deoxycholate, 200 mM NaCl) + PIC. 1% input was removed from supernatant, and 10 µg H3K4me3 (Millipore, 07-473) or 10 µg H3K27ac (Abcam, ab4729) antibody adsorbed to 60 µl ProteinG dynabeads (ab adsorbation to beads as in ChIP-seq protocol) were added to the remaining supernatant and incubated @ 4C over night with rotation. Samples were washed as follows: 2 times with 1 ml Low Salt Immune Complex Wash Buffer (0.1% SDS, 1 % Triton X-100, 2 mM EDTA, 50 mM Tris-HCl pH8, 150 mM NaCl), 2 times with 1 ml High Salt Immune Complex Wash Buffer (0.1% SDS, 1 % Triton X-100, 2 mM EDTA, 50 mM Tris-HCl pH8, 500 mM NaCl), 1 time with 1 ml LiCl Immune Complex Wash Buffer (0.25 M LiCl, 1% Igepal-CA630, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH8), 2 times with 1 ml TE buffer (10 mM Tris–HCl, pH 8.0, 10 mM EDTA) followed by elution of chromatin from magnetic beads with two rounds of 100 µl elution buffer (1%SDS, 100 mM NaHCO3) with shaking (1500 RPM) @ RT. Supernatants were transferred to new tubes and chromatin complexes were reverse cross-linked over night @ 65C with the addition of 250 mM NaCl, 100 μg RNase A (ThermoFisher, EN0531) and 50 μg proteinase K (ThermoFisher, AM2546) followed by Zymo Research ChIP DNA Clean & Concentrator (BIOSITE-D5205) clean up. 25 μL of Streptavidin T1 beads (Thermo Fisher, 65601) were washed with Tween Wash Buffer (5 mM Tris-HCl pH 8, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20) then resuspended in 50 μL of 2X Biotin Binding Buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2M NaCl). Beads were added to the samples and incubated at room temperature for 15 minutes with shaking. After capture, beads were placed on a magnet and supernatant was discarded. Samples were washed twice by adding 500 μL of Tween Wash Buffer and incubated at 55°C for 2 minutes shaking followed by a 1 time wash in 100 μl 1X T4 DNA ligation buffer. Beads were collected on a magnet and resuspended on 100 µl end-repair mastermix (0.5 mM dNTPs(VWR, E636-40UMOLE), 12U T4 DNA Polymerase (NEB, M0203), 50U T4 Polynucleotide Kinase (NEB, M0201), 5U Klenow (NEB, M0210), 1X NEB T4 DNA ligase buffer) followed by incubation @ RT for 30 min with shaking (900 RPM). 300 μl Tween Wash Buffer was added and beads were placed on a magnet and supernatant was discarded. Samples were washed twice by adding 500 μL of Tween Wash Buffer and incubated at 55°C for 2 minutes shaking followed by a 1 time wash in 100 µl 1X NEB2 buffer. Beads were collected on a magnet and resuspended on 100 µl A-tailing mastermix (0.5 mM dATP (ThermoFisher), 25U Klenow Exo- (NEB, M0212), 1X NEB2 buffer) and incubated @ 37°C for 30 min with shaking (900 RPM). 300 µl Tween Wash Buffer was added and beads were placed on a magnet and supernatant was discarded. Samples were washed twice by adding 500 μL of Tween Wash Buffer and incubated at 55°C for 2 minutes shaking followed by a 1 time wash in 100 µl 1X Fast-Link ligation buffer (Epicenter, LK0750H ) followed by NEXTflex DNA barcode ligation  (BIOO scientific) using the Fast-link ligation kit (Epicenter, LK0750H). 300 µl Tween Wash Buffer was added and beads were placed on a magnet and supernatant was discarded. Samples were washed twice by adding 500 μL of Tween Wash Buffer and incubated at 55°C for 2 minutes shaking followed by a 1 time wash in 100 µl 10mM Tris-HCl pH 8.0, resuspended in 45 µl and a 1:1000 dilution was made for qPCR determination of number cycles needed for final PCR amplification. After final PCR, T1 streptavidin beads were collected on a magnet, supernatant transferred to new tubes and Ampure XP beads (x0.8 sample volume) were used to clean up libraries. PLAC-seq libraries were subject to 2X 75 cycles of paired end sequencing on a NextSeq500.
identifiers.namespace: GEO
identifiers.id: GSM3595849