Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
Epitope tags

Cell type

Cell type Class
Lung
Cell type
MRC-5
Primary Tissue
Lung
Tissue Diagnosis
Normal

Attributes by original data submitter

Sample

source_name
Fetal Lung Fibroblast
cell line
MRC-5 (ATCC, CCL-171)
antibody
Rabbit anti-HA polyclonal antibody (Abcam, AB9110)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
(Histone 3 lysine 27 acetylation) MRC-5 fibroblasts were cultured, transduced with NEUROG2-encoding lentivirus, and treated with neuron induction medium or neuron induction medium supplemented with 10 μM forskolin and 1 μM dorsomorphin for 2 days. Approximately 1×10^7 cells were treated with Accutase cell detachment solution (Innovative Cell Technologies) for 3 minutes and collected in ice-cold PBS by centrifugation (525 × g, 5 minutes, 4°C). Cells were resuspended by repeated gentle pipetting in 4 ml ice-cold resuspension buffer (PBS containing 25 mM HEPES (pH 7.0) and 5 mM EDTA). GFP expressing viable cells were isolated using fluorescence-based sorting on a MoFlo platform (Beckman Coulter). Approximately 500,000 cells were collected by centrifugation (525 × g, 5 minutes, 4°C) then washed with 1 ml ice-cold PBS for chromatin digestion (Gilfillan et al., 2012). Cells were resuspended in 95 μl Micrococcal Nuclease digestion buffer (New England Biolabs, B0247S) supplemented with 0.01 μg BSA (New England Biolabs, B9001S), 0.2% (v/v) Triton X 100, and 1% (v/v) cOmplete EDTA-free Protease Inhibitor Cocktail. Resuspended cells were treated with 100 gel units Micrococcal Nuclease (New England Biolabs, M0247S) at 37°C for 5 minutes. Cells were immediately transferred to ice, treated with 10 μl ice-cold quench buffer (100 mM HEPES (pH 8.0) and 55 mM EDTA), and sonicated in a 1.5 ml TPX microtube for 60 seconds (power setting: high) using a Bioruptor equipped with a 4°C refrigerated water bath. Chromatin was diluted with 110 μl ice-cold immunoprecipitation buffer (50 mM HEPES (pH 8.0), 40 mM NaCl, 5 mM EDTA (pH 8.0), 0.2% (v/v) Triton X-100, 0.2% (w/v) SDS, and 1% (v/v) cOmplete EDTA-free Protease Inhibitor Cocktail), cell debris removed by centrifugation (21,130 × g, 10 minutes, 4°C), and 100 μl of supernatant transferred into two 0.2 ml microtubes (VWR, 732-0547). Chromatin was incubated with 1 μg rabbit polyclonal histone H3 acetyl K27 (Abcam, ab4729) antibody for 18 hours at 4°C with gentle nutation. The remaining 20 μl of supernatant was stored at 20°C as input. The following day, 20 μl Magnetic Protein G Dynabeads were washed three times with 200 μl ice-cold PBS containing 0.2% (v/v) Tween 20 on a MagneSphere separation stand at 4°C. The wash solution was removed and the beads were resuspended in 10 μl immunoprecipitation buffer then added to immunopreciptation samples for 2 hours at 4°C with gentle nutation. The bead complexes were magnetically isolated, twice washed by resuspension in 150 μl ice-cold immunoprecipitation buffer for 2 minutes, twice washed by resuspension in 150 μl ice-cold wash buffer (100 mM Tris-HCl (pH 9.0), 500 mM LiCl, 1% (v/v) IGEPAL CA-630, 1% (w/v) deoxycholic acid, and 1% (v/v) cOmplete EDTA free Protease Inhibitor Cocktail) for 1 minute, and finally resuspended in 150 μl ice-cold high salt wash buffer (wash buffer containing 150 mM NaCl) for transfer to a 1.5 ml microtube on ice followed by bead collection. Chromatin was eluted from collected beads in 50 μl elution buffer (1% (w/v) SDS and 50 mM NaHCO3) by two sequential additions of 25 µl elution buffer for 30 minutes at 27°C with vigorous agitation. Beads were magnetically collected and eluted chromatin was transferred to a sterile 1.5 ml microtube. Input chromatin was thawed on ice and treated with 50 μl elution buffer for 30 minutes at 27°C. Input and immunoprecipitated chromatin were sequentially treated with 10 μg RNase A for 15 minutes at 37°C and 80 μg Proteinase K for 3 hours at 55°C. Chromatin was purified with a QIAquick PCR purification kit and concentration was determined using a Qubit fluorometer and Qubit dsDNA HS assay kit as described above. (Histone 3 lysine 27 methylation) MRC-5 fibroblasts were cultured, transduced with NEUROG2-encoding lentivirus, and treated with neuron induction medium or neuron induction medium supplemented with 10 μM forskolin and 1 μM dorsomorphin for 2 days. Approximately 1×10^7 cells were treated with Accutase cell detachment solution (Innovative Cell Technologies) for 3 minutes and collected in ice-cold PBS by centrifugation (525 × g, 5 minutes, 4°C). Cells were resuspended by repeated gentle pipetting in 4 ml ice-cold resuspension buffer (PBS containing 25 mM HEPES (pH 7.0) and 5 mM EDTA). GFP expressing viable cells were isolated using fluorescence-based sorting on a MoFlo platform (Beckman Coulter). Approximately 500,000 cells were collected by centrifugation (525 × g, 5 minutes, 4°C) then washed with 1 ml ice-cold PBS for chromatin digestion (Gilfillan et al., 2012). Cells were resuspended in 95 μl Micrococcal Nuclease digestion buffer (New England Biolabs, B0247S) supplemented with 0.01 μg BSA (New England Biolabs, B9001S), 0.2% (v/v) Triton X 100, and 1% (v/v) cOmplete EDTA-free Protease Inhibitor Cocktail. Resuspended cells were treated with 100 gel units Micrococcal Nuclease (New England Biolabs, M0247S) at 37°C for 5 minutes. Cells were immediately transferred to ice, treated with 10 μl ice-cold quench buffer (100 mM HEPES (pH 8.0) and 55 mM EDTA), and sonicated in a 1.5 ml TPX microtube for 60 seconds (power setting: high) using a Bioruptor equipped with a 4°C refrigerated water bath. Chromatin was diluted with 110 μl ice-cold immunoprecipitation buffer (50 mM HEPES (pH 8.0), 40 mM NaCl, 5 mM EDTA (pH 8.0), 0.2% (v/v) Triton X-100, 0.2% (w/v) SDS, and 1% (v/v) cOmplete EDTA-free Protease Inhibitor Cocktail), cell debris removed by centrifugation (21,130 × g, 10 minutes, 4°C), and 100 μl of supernatant transferred into two 0.2 ml microtubes (VWR, 732-0547). Chromatin was incubated with 1 μg mouse monoclonal histone H3 tri-methyl K27 (Abcam, ab6002) antibody for 18 hours at 4°C with gentle nutation. The remaining 20 μl of supernatant was stored at 20°C as input. The following day, 20 μl Magnetic Protein G Dynabeads were washed three times with 200 μl ice-cold PBS containing 0.2% (v/v) Tween 20 on a MagneSphere separation stand at 4°C. The wash solution was removed and the beads were resuspended in 10 μl immunoprecipitation buffer then added to immunopreciptation samples for 2 hours at 4°C with gentle nutation. The bead complexes were magnetically isolated, twice washed by resuspension in 150 μl ice-cold immunoprecipitation buffer for 2 minutes, twice washed by resuspension in 150 μl ice-cold wash buffer (100 mM Tris-HCl (pH 9.0), 500 mM LiCl, 1% (v/v) IGEPAL CA-630, 1% (w/v) deoxycholic acid, and 1% (v/v) cOmplete EDTA free Protease Inhibitor Cocktail) for 1 minute, and finally resuspended in 150 μl ice-cold high salt wash buffer (wash buffer containing 150 mM NaCl) for transfer to a 1.5 ml microtube on ice followed by bead collection. Chromatin was eluted from collected beads in 50 μl elution buffer (1% (w/v) SDS and 50 mM NaHCO3) by two sequential additions of 25 µl elution buffer for 30 minutes at 27°C with vigorous agitation. Beads were magnetically collected and eluted chromatin was transferred to a sterile 1.5 ml microtube. Input chromatin was thawed on ice and treated with 50 μl elution buffer for 30 minutes at 27°C. Input and immunoprecipitated chromatin were sequentially treated with 10 μg RNase A for 15 minutes at 37°C and 80 μg Proteinase K for 3 hours at 55°C. Chromatin was purified with a QIAquick PCR purification kit and concentration was determined using a Qubit fluorometer and Qubit dsDNA HS assay kit as described above. (NEUROG2) MRC-5 fibroblasts were cultured, transduced with NEUROG2+HA-SOX4-encoding lentivirus, and treated with neuron induction medium or neuron induction medium supplemented with 10 μM forskolin and 1 μM dorsomorphin along the above described time course. Approximately 1×10^7 fibroblasts were treated with 16% (w/v) methanol-free formaldehyde (Thermo Scientific, 28908) at a final concentration of 1% (v/v) for 8 minutes at room temperature with gentle rotation following 24 hours in neuron induction medium. Cr

Sequencing Platform

instrument_model
Illumina HiSeq 2500

hg38

Number of total reads
43250262
Reads aligned (%)
93.9
Duplicates removed (%)
30.5
Number of peaks
20381 (qval < 1E-05)

hg19

Number of total reads
43250262
Reads aligned (%)
93.6
Duplicates removed (%)
30.9
Number of peaks
20399 (qval < 1E-05)

Base call quality data from DBCLS SRA