GSM3889217: HCT116_KAP1_KO2_Rep2_RNASeq; Homo sapiens; ssRNA-seq
For ChIP-seq, chromatin was cross-linked with 0.5% formaldehyde for 10 min at room temperature in culture media, and the reaction was stopped by the addition of glycine (150 mM final). Cells were washed twice with PBS and re-suspended in Farnham Lysis Buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 1 mM PMSF, protease inhibitor (Roche)) at 10x106 [10 million cells/mL], dounced 15X, and incubated for 30 min at 4°C. Cell nuclei were collected by centrifugation at 1000xg for 5 min at 4°C, and re-suspended in Szak's RIPA buffer (50 mM Tris-HCl pH 8.0, 1% NP-40, 150 mM NaCl, 0.5% deoxycholate, 0.1% SDS, 5 mM EDTA, 0.5 mM PMSF, protease inhibitor) at 25x106 nuclei/mL. Nuclear pellets were sonicated using the Bioruptor water bath sonicator (Diagenode) using the high setting with 30 sec on 30 sec off for 45 min to generate 100-300 bp DNA fragments. Approximately 20x106 nuclei were used for each ChIP. For the ChIP step, 5-10 ug of antibody was conjugated with 100 uL of protein G dynabeads Thermo Fisher and blocked with 5% bovine serum albumin in RIPA for 1 hr at 4°C. An additional 2.5 µg of antibody specific for Drosophilla H2Av (Active Motif, 61686) was also conjugated at the same time. Drosophilla spike-in chromatin (Active Motif, 53083) was added to each sample for normalization between samples according to manufacture's instructions. Antibody-conjugated dynabeads were incubated with sheared chromatin suspension overnight at 4°C. Then the beads were sequentially washed two times with each of the following buffers: Szak's RIPA buffer (see above), low salt buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton, 0.1% SDS, 2 mM EDTA), high salt buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 1% Triton, 0.1% SDS, 2 mM EDTA), LiCl wash buffer (0.25M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 20 mM Tris, pH 8.0), and 1 mM Tris-EDTA pH 8.0. Chromatin immunocomplexes were eluted by incubation for 30 min at 65°C with 1% SDS and 100 mM NaHCO3, and cross-linking was reversed by incubation in the solution adjusted to 200 mM NaCl and proteinase K (20 μg) for 1 hr at 65°C. DNA was then purified using the Zymo ChIP DNA Clean & Concentrator Kit (Zymo Research). Final ChIP DNA was quantified on a Qubit 2.0 Fluorometer (Invitrogen) and approximately 5-10 ng of DNA were submitted (used?) for library preparation.  For 4sU-seq, HCT116 Ctrl and KAP1 KO1 cells were seeded into three 10cm plates per biological replicate and grown to 70% confluency. Cells were pulsed for 10 min with DMEM + 500 µM 4sU (Sigma-Aldrich, T4509). At the 9 min mark the DMEM + 4sU media was removed and cells were washed one time with 1X PBS. The PBS was removed, and cells were lysed on-plate using the standard TRizol protocol (ThermoFisher Scientific, 15596026). RNA samples were DNase treated (NEB, M0303) and then extracted with acid:phenol chloroform (ThermoFisher Scientific, AM9720) followed by an additional chloroform extraction. RNA was then precipitated with 1/10 volume of NaCl (5M), 1 µL glycoblue, and 2.5 volumes of 100% EtOH. RNA pellets were washed with 1 mL of 75% EtOH and air dried. RNA pellets were then resuspended in RNase-free H2O. RNA (300 ug) was brought to a final concentration of 0.4 mg/mL with RNase-free H2O. A separate aqueous master mix (20mM NaOAC pH 5.2, 1 mM EDTA pH 8.0, 0.1% SDS) was added to the diluted RNA mix followed by the addition of 0.2 mg/mL Biotin-HPDP (ThermoFisher Scientific 21341) in DMF. The biotinylation reaction was incubated at room temperature for 2 hrs followed by acid:phenol chloroform extraction. Biotinylated RNA pellets were resuspended in 500 µL RPB buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 300 mM NaCl). Dyna MyOne Strepavidin T1 beads (200 uL/sample; ThermoFisher Scientific, 65601) were blocked at room temperature in beads wash buffer + 1% polyvinylpyrrolidone for 10 min. After blocking, beads were washed 1X with 1 mL beads wash (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 50 mM NaCl) buffer and then resuspended in RPB. Biotinylated, 4sU RNA was denatured at 65°C for 5 min and then placed on ice for 2 min. 200 µL of blocked beads were added to each sample and incubated for 30 min with rotation at room temperature. Beads were washed 5X with 4sU wash buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 1 M NaCl, 0.1% Tween-20). Biotinylated, 4sU RNA was eluted from beads 2 times in 75 µL of 0.1M DTT for 15 min at room temp with rotation, giving a final elution valume of 150 µL. 4sU-RNA was further purified using the Zymo RNA Clean and Concentrator Kit (Zymo Reasearch, R1013). RNA concentration was measured using the Qubit RNA HS assay (ThermoFisher Scientific, Q32852) and quality was determined using the Agilent Tapestation RNA ScreenTape (Agilent, 5067-5576).  For RNA-seq, HCT116 Ctrl and KAP1 gRNA cells were grown in 6-well dishes and total RNA extracted using the Zymo Quick-RNA Miniprep Kit (R1055) according to manufacturer instructions.  For MNase-seq, cells were crosslinked with 0.5% methanol-free formaldehyde for 10 min and reaction was quenched for 5 min with 150 mM glycine before harvesting. Cells were washed twice with PBS buffer and nuclei were gathered by lysing the cells in Franham's lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl and 0.5% NP-40 freshly supplemented with 1mM PMSF and EDTA free-Protease inhibitor cocktail). Nuclei were washed once with cold MNase buffer (20 mM Tris-HCl pH 7.5,15 mM NaCl, 60 mM KCl, 2 mM CaCl2) and resuspended in MNase buffer with concentration of 10x106 cells/mL before digestion. Micrococcal nuclease (New England BioLabs, M0247S) digestion was performed (240 U/106 cells) at 37C to achieve 80% mono-nucleosomes populations and reaction was stopped with 10 mM EDTA, 20 mM EGTA, 0.4% SDS solution. Samples were centrifuged for 5 min at high speed and supernatant was collected. Samples were reverse crosslinked in de-crosslinking buffer (20 mM Tris- HCl pH 6.8, 500 mM NaCl, 2mM EDTA, and 0.5 mg/mL Proteinase K) overnight at 65°C. Following phenol-chloroform extraction and ethanol precipitation, DNA samples were run on 1.5% agarose gel and fragments of ~150 bp size were excised and purified. DNA from two biological replicates were analyzed on high sensitivity DNA tape on a 2200 TapeStation and used for library preparation. For ChIP-seq and MNase-seq, libraries were constructed using the KAPA Hyper Prep Kit (KAPA Biosystems, KK8504) according to manufacturer's instructions. Brielfy, 5-10 ng of either ChIP or MNase digested DNA was end repaired and A-tailed. Illumina adapters (New England BioLabs, E7335L) were then ligated to DNA fragments. ChIP DNA was size selected using the double-sided selection method outlined in the KAPA Hyper Prep Kit instructions. DNA was then amplified using Illumina indexed barcoding primers (New England BioLabs, E7335L) for 8-12 cycles depending on amount of input DNA.  For 4sU-seq and RNA-seq, the KAPA Stranded RNA-seq kit with RiboErase (HMR) (KAPA Biosystems, KK8483) was used to prepare libraries according to manufacturer's instructions. Briefly, 200 ng of 4sU RNA and 1 ug of total RNA was used to prepare 4sU-seq and RNA-seq libraries, respectively. Libraries were rRNA depleted followed by DNase treatment. RNA was fragmented and primed followed by 1st strand cDNA synthesis. 2nd strand synthesis was then performed with the incorporation of dUTP. Libraries were then A-tailed, adapter ligated, and amplified as described for ChIP-seq.  All libraries were quantified using the Qubit 2.0 fluorometer and library size verified using the Agilent Tapestation DNA D1000 ScreenTape (Agilent, 5067-5582). All libraries were submitted to the McDermott Center Sequencing Core at UTSW. 75 bp paired-end reads were generated for all libraries