GSM3721079: Pool2; Homo sapiens; RNA-Seq
Proximity RNA-Seq    Frozen cell pellets from single 150 mm dishes were thawed on ice in 1 ml 20 mM Tris pH 7.2, spun and resuspended in 1.5 ml hypotonic Igepal C-630 lysis buffer (20 mM Tris, pH 7.2, 5 mM NaCl, 0.2% Igepal C-630, 2 mM EDTA, 1 mM EGTA) supplemented with 1x Complete, EDTA-free protease inhibitor cocktail (Roche Applied Science) and 0.5 units/µl SUPERase IN RNase inhibitor (Thermo Fisher Scientific). Cells were kept on ice for 30 min with occasional mixing. Nuclei were spun at 2 krpm for 5 min in a benchtop centrifuge at room temperature, and the buffer exchanged with SDS washing buffer (20 mM Tris, pH 7.2, 5 mM NaCl, 0.3% SDS, 2 mM EDTA, 1 mM EGTA) supplemented with inhibitors as listed for the hypotonic Igepal C-630 lysis buffer. Samples were incubated for 20 min at room temperature in a thermoblock with constant mixing at 750 rpm. After 10 min of the incubation, Triton X-100 was added for a final concentration of 1.7%. Nuclei were then washed once in 10 mM Tris pH 7.2, 5 mM NaCl, 0.5 mM EDTA, 1% Triton X-100, supplemented with inhibitors as specified above, and once in the same buffer with only 0.05% Triton X-100. Nuclei were resuspend in 0.2 ml wash buffer with 0.05% Triton X-100 and sonicated in 15 ml falcon tubes using a bioruptor UCD-200 sonicator (Diagenode) with power set to medium for 3-5 cycles of 10 seconds on followed by 10 seconds off at 8C. After 3 cycles of sonication, the disruption of nuclei was inspected using trypan blue staining and light microscopy. Sonicated nuclear homogenates were flash frozen in liquid nitrogen and stored at -80C until use.     Hi-C    Nuclei were prepared by lysing cells (2-10 million cells) in 10 mM Tris pH 8, 0.2 % Igepal CA-420, 10 mM NaCl supplemented with protease inhibitor cocktail tablet (Roche complete, EDTA-free) for 30 min on ice. Nuclei were pelleted at 300 x g for 5 minutes at 4C, then nuclei were washed in 1 ml ice-cold 1.25x NEbuffer 2 and resuspended in 360 µl of the same buffer. 11 µl 10% SDS per tube (0.3% final) were added, mixed carefully by pipetting, and incubated at 37°C for 45 min, shaking at 950 rpm. To disrupt remaining clumps, nuclei were pipetted up and down several times during incubation. 75 µl 10% Triton X-100 per tube (1.6% final) was added, and the mixture was incubated at 37°C for 45 min, shaking at 950 rpm Proximity RNA-Seq - further details discussed in doi: https://doi.org/10.1101/196147    Hi-C    To digest the chromatin 1500 units of HindIII (15 µl at 100U/µl NEB) were added and the reaction mix was incubated at 37°C overnight while rotating at 950 rpm. The reaction was then placed on ice. To fill in the restriction fragment overhangs and mark the DNA ends with biotin, 1.5 µl 10 mM dCTP, 1.5 µl 10 mM dGTP, 1.5 µl 10 mM dTTP, 37.5 µl 0.4 mM biotin-14-dATP (Invitrogen) and 10 μl 5 U/μl Klenow (DNA polymerase I large fragment, NEB) were added and mixed before incubation for 75 minutes at 37°C while shaking at 700 rpm every 30 seconds for a duration of 10 seconds. Repaired ends were then re-ligated. A 2x ligation mix of following composition was added: 100 µl 10x ligation buffer (NEB), 10 μl 10mg/ml BSA, 15 µl 1U/μl T4 DNA ligase (Invitrogen) and 425 µl water. The reaction was mixed by pipetting and incubated overnight at 16ºC. After pelleting at 300 x g for 2 min, nuclei were resuspended in 400 µl TLE and 50 µl 10 mg/ml Proteinase K (Roche) and incubated at 37ºC for two hours. Formaldehyde crosslinks were reversed by increasing the temperature to 65ºC for the duration of around 16 hours. Then 20 µl of 10 mg/ml RNase A were added and the mixture incubated at 37ºC for 1 hour. DNA was purified by Phenol/chloroform extraction and ethanol/Na acetate precipitation. Purified DNA was quantitated using the Quant-iT PicoGreen kit (Invitrogen) according to manufacturer's instructions. DNA was then sonicated using a Covaris LE220 sonicator. The sample (from 5 to 10 µg) was diluted in 130 µl water per tube and sonicated to 300-500 bp fragments using the following settings: fill level 10, duty cycle 15, peak incident power (w) 500, cycles per burst 200, time (seconds) 58.   Ends were repaired and accessible biotin removed by the addition of 16 µl ligation buffer (10X), 2 µl 10 mM dATP, 5 µl T4 DNA Polymerase (15 U per 5 µg HiC library, NEB), 7 µl water for a total of 160 µl. The mix was incubated at 20ºC for 30 min. Then 5 µl of 10 mM dNTPs, 4 µl 10x Ligation buffer, 5 µl T4 PNK (10 U/µl, NEB), 1 µl Klenow and 25 µl water for a total of 200 µl was added, and the mixture was incubated at 20ºC for 30 min. The reaction was terminated by purification and size selection of DNA fragments aiming at a length of around 250-550 bp using Ampure XP beads (Beckman Coulter) according to manufacturer's instructions. A first round of selection was performed with a volume of Ampure XP beads 0.6 x the DNA sample volume followed by a second round with 0.9 x the volume. DNA was finally eluted in 300 µl TLE. Ligation products with internal biotin were selected using 150 µl Dynabeads MyOne Streptavidin C1 beads (Invitrogen). Beads were washed in 400 µl 5 mM Tris pH 8.0, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween (TB) and then resuspended in 300 µl Tris-HCl pH8.0, 1mM EDTA, 2M NaCl (NTB). Beads were then mixed with Hi-C DNA and incubated for 20 min. Subsequently, beads were capture on a magnetic rack and washed twice with 5 mM Tris pH 8.0, 0.5 mM EDTA, 1M NaCl and once in 0.5 x TB for 3 min at 55ºC rotating at 750 rpm. Beads were then washed once in 1x NEB2 buffer (NEB) and resuspended in 10 µl 10 x NEB 2, 5 µl 10mM dATP, 5 µl Klenow exo- (NEB) and 80 µl water to A-tail DNA ends. The mixture was incubated for 37ºC for 30 min. Two washes followed with 400 µl 0.5x TB incubating at 55ºC for 3 min rotating at 750 rpm. After an additional wash in 1x T4 DNA ligation buffer (NEB), beads were resuspended in 50 µl 1x ligation buffer and transferred to a new tube. 4 µl of Hi-C adapter (15 µM stock) and 2 µl NEB T4 Ligase (400 U/µl, NEB) were added, and the reaction was incubated at room temperature for 2 hours. Beads were captured on the magnetic rack and then washed twice in TB, once in NTB and finally in 1x NEB2 buffer before resuspension in 40 µl 1x NEB2. Hi-C libraries were amplified by PCR off the beads using 5 µl beads in a 50 µl reaction using PCR kit (Phusion, NEB) according to manufacturer's instructions. PCR products were purified with 0.85 x volume Ampure XP beads according to manufacturer's instructions and eluted with 30 µl TLE. The concentration and fragment size distribution of libraries were determined by Bioanalyzer profiles (Agilent Technologies) and Kapa Illumina SYBR green qPCR (Kapa Biosystems) according to manufacturer's instructions.
GEO Accession
GSM3721079