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Sample GSM1482822

Query DataSets for GSM1482822

Status

Public on May 07, 2015

Title

Input_ICeChIP-seq_HEK293_2

Sample type

SRA

Source name

Cell culture

Organism

Homo sapiens

Characteristics

cell line: HEK 293
chip antibody: none
growth protocol: HEK293 cells were grown in 37°C, 5% CO2, 95% humidity in high glucose DMEM (Invitrogen) supplemented with 10% (v/v) HyClone FBS Characterized U.S. and 1X Penicillin/Streptomycin (Gibco). HEK293 cultures were grown in 15 cm polystyrene dishes, passaged 1:20 every 5 days, Cells used in ChIP experiments were harvested when 80-90% confluent with media changed at least 3 hours prior harvesting.
amplification cycles: 12
map quality cut-off: 13
mononucleosome cut-off: not performed
reference genome: hg19

Growth protocol

HEK293 cells were grown in 37°C, 5% CO2, 95% humidity in high glucose DMEM (Invitrogen) supplemented with 10% (v/v) HyClone FBS Characterized U.S. and 1X Penicillin/Streptomycin (Gibco). HEK293 cultures were grown in 15 cm polystyrene dishes, passaged 1:20 every 5 days, Cells used in ChIP experiments were harvested when 80-90% confluent with media changed at least 3 hours prior harvesting.
Mouse Embryonic Stem Cells (mESC) E14 cell line (129/Ola background) (Hooper et al., 1987) were grown at 37°C, 5% CO2, 95% humidity in ES media high glucose DMEM (Invitrogen) supplemented with 15% (v/v) FBS (Gibco), 2mM L-glutamine (Gibco), 1% (v/v) non-essential amino acids (Gibco), 1X Penicillin/Streptomycin (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), 1000U/mL LIF (ESG1107, Millipore), 3 µM CHIR99021 (04-0004, Stemgent), 1 µM PD0325901 (04-0006, Stemgent), sterile filtered 0.1 µm membrane, stored in 4°C for up to 1 week. E14 cells were cultured on dishes coated with 0.1% gelatin (Sigma) without feeder cells, and passaged daily in 1:3 ratio (media was changed 3 hours prior passage). Cells were harvested at 80-90% confluence, with media changed at least 3 hours prior.

Extracted molecule

genomic DNA

Extraction protocol

Our ICeChIP protocol is based on input preparation largely following the Dilworth lab native ChIP protocol (Brand et al., 2008). Plate-adhered cells (~107 cells) were washed twice with 10 mL of PBS, and released by 5 mL Accutase (Millipore) for 5 minutes in 37°C, quenched with 2 mL of complete media, and collected by centrifugation (500 x g, for 5 minutes at 4°C). All subsequent steps were performed on ice with ice-cold buffers. Cells were washed twice with 10 mL PBS, and twice with 5 mL of Buffer N (15 mM Tris pH 7.5, 15 mM NaCl, 60 mM KCl, 8.5% (w/v) Sucrose, 5 mM MgCl2, 1 mM CaCl2 1 mM DTT, 200 μM PMSF, 1x RL Protease Inhibitor Cocktail). Cells were resuspended in 1mL of Buffer N and lysed by adding 1 mL of 2x Lysis Buffer (Buffer N supplemented with 0.6% NP-40 substitute (Sigma)) for 10 minutes at 4°C. Nuclei were collected by centrifugation (500 x g for 5 minutes at 4°C) and were resuspended in 6 PCVs (packed cell volumes) of Buffer N. To remove cell debris, resuspended nuclei were overlaid on the surface of 7.5 mL of sucrose cushion (10 mM HEPES pH 7.9, 30%(w/v) sucrose, 1.5 mM MgCl2) in a 50 mL centrifuge tube centrifuged (1300 x g, Sorvall Legend XTR swinging bucket rotor for 12 minutes at 4°C). Most cell debris remained in upper layer while nuclei sedimented through the sucrose cushion and pelleted on the bottom of the tube. The supernatant was discarded and nuclei were resuspended in 2 PCVs of Buffer N. To measure apparent concentration of chromatin, 2 μL of resuspended nuclei were diluted in 98 μL of 2M NaCl in triplicate, total nucleic acid absorbance was measured at 260 nm by Nanodrop (Thermo Scientific), and the conversion factor assuming 1A260 = 50 ng/μL of chromatin employed. Based on these measurements, apparent concentration of chromatin was adjusted to 1 μg/μL with Buffer N. The quantity and quality of nuclei were also assessed using hemocytometer. Chromatin was sheared through exhaustive MNase digest. At this stage, a ladder of semisynthetic nucleosomes was doped into the pool of native nucleosomes. The amount of spiked ladder was comparable to estimated amount of genome copies in the pool based on the nuclei counting times the average DNA content per cell (~2.5 copy of genome per cell). To remove debris coming from nuclei lysis and MNase digestion as well as strip chromatin bound factors, the pool of nucleosomes was subjected to hydroxyapatite chromatography purification (Brand et al., 2008). Fragmented chromatin with internal standard ladders were split into 100 μg total nucleic acid fractions and each fraction was mixed with 66 mg of hydroxyapatite (HAP) resin (Bio-Rad Macro-Prep® Ceramic Hydroxyapatite Type I 20 μm) rehydrated with 200 μL of HAP buffer 1(3.42 mM Na2HPO4 and 1.58 mM NaH2PO4 final pH 7.2, 600 mM NaCl, 1 mM EDTA, 200 μM PMSF), incubated for 10 minutes at 4°C on rotator and subsequently was applied to the centrifugal filter unit (Millipore Ultrafree MC–HV Centrifugal Filter 0.45 μm). The chromatin-loaded resin in the column was drained and then washed four times with 200 μL HAP buffer 1 and four times with 200 μL of HAP buffer 2 (3.42 mM Na2HPO4 and 1.58 mM NaH2PO4 final pH 7.2, 100 mM NaCl, 1 mM EDTA, 200 μM PMSF) by centrifugation (600 x g, 1 minute at 4°C in fixed angle rotor. Nucleosomes were eluted from the HAP column with three 100 μL washes of HAP elution buffer (342 mM Na2HPO4 and 158 mM NaH2PO4 final pH 7.2, 100 mM NaCl, 1 mM EDTA, 200 μM PMSF). To measure apparent concentration of HAP purified chromatin fragments, 10 μL of HAP elution was diluted in 40 μL of 2M NaCl in triplicate, and absorbance measured at 260 nm averaged and adjusted (1A260 = 50 ng/μL of chromatin). Apparent concentration of chromatin was adjusted to 20 μg/mL with ChIP Buffer 1(25 mM Tris pH 7.5, 5 mM MgCl2, 100 mM KCl, 10% (v/v) glycerol, 0.1% (v/v) NP-40 substitute). H3K4me3 ChIP was performed with 10 μg of chromatin and 15 μL of AM39159 antibody, H3 and H4 ChIP was performed with 1 μg of chromatin and 15 μL of AM61277 and AM61299 antibody, respectively (Active Motif). 10% of initial chromatin for each IP was set aside to serve as ChIP input. Each IP experiment used 50 μL of Protein A Dynabeads (Invitrogen) that were washed twice with 1mL of ChIP buffer 1 with 1 min collection on magnetic rack after each wash. To prepare the resin, 15 μL of antibody and 85 μL of ChIP buffer 1 was added to Protein A Dynabeads and incubated for 10 minutes at room temperature on a rotator, then washed twice with 1mL of ChIP Buffer 1. Chromatin (10 μg unless otherwise indicated) in 500 μL of ChIP buffer 1 was then added to magnetic beads and incubated for 15 minutes at room temperature on rotator. Beads were washed 3 times with 1 mL of ChIP Buffer 2 (mM Tris pH 7.5, 5 mM MgCl2, 300 mM KCl, 10% (v/v) glycerol, 0.1% (v/v) NP-40 substitute), then twice with ChIP buffer 3 (10 mM Tris pH 7.5, 250 mM LiCl, 1mM EDTA, 0.5% Na•Deoxycholate, 0.5%(v/v) NP 40 substitute), each wash consisting of a 10 minute rotating incubation and 1 minute collection on magnetic rack at 4ºC. During the course of washing, at least two tube changes reduced non-specific background. Beads were then rinsed with 1 mL of ChIP Buffer 1 and 1 mL of TE buffer, followed with two 200 μL ChIP elution buffer steps (50 mM Tris pH 7.5, 1 mM EDTA, 1% w/v SDS). Each elution step consisted of 10 minute incubation at 65ºC in a Thermoshaker (Eppendorf) at 900 rpm. Elutions were combined and ChIP elution buffer was added to inputs to match volume of ChIP elutions. After adjusting the buffer to 200mM NaCl, 100 ng of RNase A was added into the mixture and incubated at 65ºC for 45 minutes in Thermoshaker at 800 rpm, and terminated with 10 mM EDTA. Next, protein digestion was accomplished with 20 ug of proteinase K (Roche) for 2 hrs at 42ºC in the Thermoshaker at 800 rpm. DNA was recovered and purified with Qiaquick columns (Qiagen): 6 volumes of PB buffer were added to the digestion and this solution applied to the column (17900 x g, 30s) followed by 3x 750 μL of PE buffer washes (17900 x g, 30s) with an extra 1 minute spin to remove residual ethanol. DNA was eluted by applying two times 25 μL of TE buffer at 50ºC and centrifuging (17900 x g, 1 min).
For library preparation 10 ng of DNA isolated from IP or input was used. In cases in which the total amount of DNA was below 10 ng, all available DNA was subjected to library preparation. Ends of DNA were blunted using the End-it™DNA End-Repair Kit (Epicentre) (7μL 10x End-It buffer, 7μL 2.5 mM dNTP Mix, 7μL 10 mM ATP, 1.4μL of End-Repair Enzyme Mix and 47.6μL of DNA in TE buffer, incubated for 45 minutes at room temperature. DNA was purified with 126μL (1.8 volume) of Ampure XP Beads (Beckman Coulter). Beads were mixed with End repair mixture by pipetting 10 times up and down followed by 5 minutes incubation at room temperature. Magnetic beads were collected on side of the tube by magnet and two 30 sec 250 μL 80% EtOH washes on magnet were performed. Tubes were removed from the magnetic rack and 34 μL of TE buffer was added to beads and pipetted 10 times up and down. Magnetic beads were not removed from elution and remained in the tube during A-tailing. Addition of single adenosine to 3’ ends of DNA was accomplished by adding to 5 μL NEB buffer 2, 10 μL 1 mM dATP, 1 μL Klenow fragment (3’→5’ exo-, NEB) to the End-repaired DNA, and with incubation at 37ºC for 30 minutes. To purify DNA, 110 μL (2.2 volume) of SPRI Buffer (20% PEG6000, 2.5M NaCl) was added to the reaction and was pipetted 10 times up and down followed by 5 minutes incubation at room temperature. Magnetic beads were collected on side of the tube with magnet and two 30 sec 200 μL 80% EtOH washes on magnet were performed. Tubes were then taken out of magnetic rack and 13 μL of TE buffer was added to beads and mixed by pipette. Magnetic beads were not removed from elution and remained in the tube during adaptor liga

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Description

Sample 6
Samples 5, 6 make a set. It was calibrated using First generation barcodes (601_0708, 601_1920, 601_0102, 601_0916, 601_2122, 601_2324)
Processed data file(s):
AR5_INPUT_mQ0_e150(Genome_Coverage).bigwig
AR5_IP_INPUT_L0_mQ0_c0_e150_HMD.bigwig

Data processing

Data analysis was performed with Galaxy (Blankenberg et al., 2010; Giardine et al., 2005; Goecks et al., 2010)
Raw reads in FastQ fromat were first submitted to FastQ Groomer
Reads were mapped with Bowtie2 (Langmead et al., 2009) (sensitive preset option, end-to-end alignment), depending on organism of origin, to human (hg19) or mouse (mm10 or mm9) or fly (dm3) reference genomes with sequences of barcodes catenated at the end (each barcode with its own entry)
Resulting SAM files were then filtered using SAMtools (Li et al., 2009). Reads that were unmapped, unpaired (distance >1000 bp) and paired in wrong pair were removed from the set by this data analysis pipeline.
To remove noise coming from low quality reads and contaminants as well to mask repeatable genomic sequences, reads with mapping quality lower than 20 (mQ20) or 13 (mQ13) were removed.
To avoid signal artifacts and not distort Poisson sampling statistics paired reads were merged together into single entries (overlapping fragments were flattened and gaps were filled). To avoid oligonucleosome avidity bias, reads longer than 220 bps (L0-220) or 200bps (L200) or 190 bps (L190) were removed, except where explicitly stated otherwise (L0). When indicated single end reads were extended to the length of 150 bps (e150).
In order to compute barcode IP enrichment, we calculated the ratio of integrated coverage over the whole sequence of each baracode in IP over the input. To calculate Histone Modification Density (HMD) we have divided genome coverage over 1-bp window for IP by genome coverage over 1-bp window in the input, this value was subsequently divided by average barcode IP enrichment and multiplied by 100%.
BEDTools (Quinlan and Hall, 2010) were used to create genome coverage bedgraphs.
Genome_build: hg19, mm10, mm10, dm3
Supplementary_files_format_and_content: bigwig (Score). Score: HMD - histone modification density; min95_CI_HMD - Lower confidence interval for HMD; max95_CI_HMD - Higher confidence interval for HMD; (Genome_Coverage) - integrated reads depth over 1-bp window; (K4_K9_K27) - HMD linearly decovoluted from H3K4me3, H3K9me3 and H3K27me3); (K4_K9_K27_K36_K79) - HMD linearly deconvoluted from H3K4me3, H3K9me3, H3K27me3, H3K36me3, H3K79me3)

Submission date

Aug 21, 2014

Last update date

May 15, 2019

Contact name

Alexander Ruthenburg

Organization name

The University of Chicago

Department

Molecular Genetics and Cell Biology

Lab

Ruthenburg Laboratory of Chromatin Biochemistry & Structure