GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM1376182

Query DataSets for GSM1376182

Status

Public on Mar 24, 2015

Title

Hela_digest_Input

Sample type

SRA

Source name

Hela

Organism

Homo sapiens

Characteristics

cell type: CCL-2
passages: NA
chip-antibody: none

Treatment protocol

Cells were cultured as described under growth protocol, to confluency.

Growth protocol

Cells were cultured in MEM containing Glutamax (Gibco), 1% non-essential amino acids, 10% fetal calf serum and 1% penicillin/streptomycin.

Extracted molecule

genomic DNA

Extraction protocol

Sonication: Cells (10e7 per ChIP) were cross-linked in suspension for 10 min in PBS containing 1% formaldehyde before quenching with 1.25 mM glycine. Cells were lysed for 30 min at 4oC on a rotator in RIPA buffer (140 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 1 mM PMSF, 1x protease inhibitor cocktail) adjusted to 1% SDS, and sonicated for 3 times 15 min in a Bioruptor (Diagenode) with 30 sec ON/OFF at high power to generate chromatin fragments of ~200-400 base pairs (bp). After sedimentation, chromatin (supernatant from 107 cell-equivalents) was diluted 10 times in RIPA buffer without SDS, and incubated on a rotator overnight at 4oC with 50 µg anti-lamin A/C antibody (Santa Cruz sc-7292) pre-coupled to magnetic Dynabeads Protein G (Invitrogen). An irrelevant mouse IgG was used as control. ChIP material was collected and washed 3 times in 1 ml of ice-cold RIPA buffer without protease inhibitors. The crosslink was reversed and DNA eluted for 6 h on a shaker at 37oC in elution buffer (50 mM NaCl, 20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% SDS) containing 0.5 µg/ml RNase A and 2 µg/ml Proteinase K). DNA was purified as described, processed for library preparation.
Chromatin preparation by micrococcal nuclease (MNase) digestion: For chromatin preparation by MNase, cells were fixed with 1% formaldehyde for 10 min, washed and formaldehyde quenched with 125 mM glycine for 5 min. After a wash in PBS, cells were scrapped in cold PBS containing 0.5 mM PMSF, sedimented and suspended in hypotonic buffer (10 mM Tris pH 7.5, 5 mM NaCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.5 mM PMSF, protease inhibitors) for 30 min on ice before Dounce homogenization (50 strokes). MgCl2 was adjusted to 5 mM and NP-40 added to 0.1%. The lysate was kept on ice for 10 min before centrifugation at 2400 g for 10 min. The pellet was resuspended in MNase buffer (20 mM Tris pH 7.5, 15 mM NaCl, 1 mM CaCl2) at 25x106 cells/ml and pre-incubated at 37oC for 5 min before adding 0.37 U MNase (Sigma; N5386) per 106 cells. Incubation was at 37°C for 5 min and digestion stopped with 10 mM EDTA on ice for 10 min. Samples were diluted to 8x10E6 cells/ml with lysis buffer (50 mM Tris pH 8.0, 10 mM EDTA, 0.5% SDS, 0.5 mM PMSF and 1x protease inhibitors). Release of digested chromatin and lamins was facilitated by pulse-sonication with a 3 mm probe for 6 times 10 sec (1 sec on/off) at 20% amplitude with cooling on ice between each round (see Results). Samples were centrifuged at 10,000 g for 10 min at 4°C and the supernatants (chromatin fraction) collected. Chromatin was then diluted 5x in NP-40 buffer (20 mM Tris pH 8.0, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 1x protease inhibitors) without SDS to decrease SDS concentration to 0.1%. Chromatin was pre-cleared for 1 h at 4oC with Protein A/G agarose beads (Santa Cruz sc-2003) and incubated overnight at 4oC with the same anti-lamin A/C antibody as above (6 µg for 6x106 cells) or with anti-lamin B1 antibodies (Abcam ab16048; 3 µg for 6x10E6 cells). Immune complexes were collected using Protein A/G agarose beads for 2 h on a rotator at 4°C, washed 6x 5 min on a rotator at 4°C as follows: once in 20 mM Tris pH 8.0, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% SDS, protease inhibitors; twice in 20 mM Tris pH 8.0, 5 mM EDTA, 500 mM NaCl, 1% NP-40, 0.1% SDS, protease inhibitors; twice in 10 mM Tris pH 8.0, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% deoxycholate, protease inhibitors; and once in TE buffer. Note that the NaCl- and LiCl-based washes used here are recommended in ChIP protocols using agarose beads. Crosslink was reversed and DNA was eluted and purified as above. Library preparation and sequencing were done as above.
The sequencing library was prepared according to the Illumina protocol for the HSeq2500 at the Norwegian Sequencing Center.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Data processing

ChIP-seq reads were aligned to the hg19 reference genome using bowtie2 version 2.1.0
Duplicate reads were removed using picard MarkDuplicates
ChIP and Input experiments were ensured to have equal read depth by downsampling the deeper sequenced experiment using picard DownsampleSam
Peak calling was done using EDD version 1.1
Genome_build: HG19
Supplementary_files_format_and_content: bed

Submission date

Apr 29, 2014

Last update date

May 15, 2019

Contact name

Philippe Collas

Organization name

University of Oslo

Department

Institute of Basic Medical Sciences

Street address

PO Box 1112 Blindern