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Sample GSM1294941

Query DataSets for GSM1294941

Status

Public on Feb 01, 2014

Title

CBX4 C-terminal input

Sample type

SRA

Source name

293T Rex cell line

Organism

Homo sapiens

Characteristics

pull-down biotap: none

Treatment protocol

Harvested 293 T-REx cells were homogenized by using a 100 ml Dounce homogenizer (Bellco, Glass Inc.), 10 strokes of each of A and B pestles. For every 4-5 ml of cell pellet volume 100 ml of NEB buffer+0.1 mM PMSF pre-chilled on ice were added. Without delay, 100 ml of cell/nuclear homogenate was poured into a T-225 flask containing a room-temperature mixture of 360 ml of PBS and 40 ml of 37% formaldehyde and incubated for 30 min at 25°C on a orbital shaker platform with vigorous shaking (100rpm). Fixed nuclei were pelleted by spinning for 10 min at 4000 g, +4°C. The supernatant was carefully decanted and the nuclear pellet was washed four times with 100 ml of ice-cold PBS with 0.1 mM PMSF. Nuclei were pelleted between washes for 10 min at 4000g, +4°C. Nuclei were resuspended in glycerol buffer and snap-frozen in liquid N2 prior to further processing. Frozen nuclear extracts from human 293 T-Rex cells were thawed and spun down for 10 min at 4000 g, +4°C. Pellets were washed with 10-20 volumes of TE buffer with 0.1 mM PMSF and spun down for 10 min at 4000g, +4°C. Pellets were again re-suspended with 10 volumes of TE buffer with 0.1 mM PMSF by pipetting up and down. SDS was added to the mixture to a final concentration of 1%. The mixture was inverted in the tube 10 times and spun down for10 min at 4000 g, +4°C. The supernatant was carefully removed (Note: the pellet may be quite loose) and the pellet was re-suspended with 10 volumes of TE buffer with 0.1 mM PMSF by pipetting and further spun down for 10 min at 4000 g, +4°C. This washing step was repeated twice. The pellet was re-suspended with 1.5 volumes of TE buffer with 0.1 mM PMSF by pipetting up and down. SDS was added to a mixture to a final concentration of 0.1%. The resulting viscous mixture was sonicated in 4.5 ml aliquots using a Misonix Sonicator 3000 with Microtip power output level 7 and total sonication processing time of 5 min (human sample), 15 sec pulse “on” and 45 sec “off” time to generate DNA fragments in the range of 300-2000 bp. Triton X-100 (1% final) and NaCl (140 mM final) were added to the sonicated samples. Samples were mixed on a rotating wheel for 5 min at +4C and spun down for 10 min at 10000 g, +4°C. The supernatant containing the soluble chromatin was collected. Note: In order to control for chromatin input composition and quality (for protein, DNA and RNA), a 1 ml aliquot is reserved before proceeding with the next step.

Growth protocol

Human 293 T-REx cells were grown in 150mm x 25mm dishes in DMEM (Invitrogen Cat #11965), supplemented with 10% FBS, 1% penicillin/streptomycin. 1.5 × 109 cells were harvested. Cell cultures were split in half by adding an equal volume of HyClone CCM3 serum-free medium (Thermo Scientific, Cat No.SH30065) and grown in eight T-flasks (225 cm2) to a density of ~5x106 cells/ml. Cells were transferred into four 2.8L Fernbach glass flasks (Bellco, Glass Inc.).

Extracted molecule

genomic DNA

Extraction protocol

Soluble chromatin was incubated with IgG agarose beads (Sigma, Cat # A2909). For every 10 ml of sonicated chromatin, 0.5-1 ml beads were added. The mixture was rotated end-over end in the 15 ml Falcon tubes for 12-16 h at +4°C. Beads were then washed 3 times for 10 min at +4°C with 15 bead volumes of RIPA buffer and spun down for 5 min at 1000g, +4°C between washes. Beads were then washed for 10 min with 15 bead volumes of buffer TEN 140 buffer with 0.1% Triton X-100 at +25°C and spun down for 5 min at 1000 g, +25°C. In order to elute the complexes (protein-DNA-RNA), 12-15 bead volumes of IgG Elution buffer were added to the beads. The slurry was mixed by inversion for 1 hour at +25°C. This elution step was repeated one more time. In order to eliminate urea from the samples, the eluates were first concentrated in 10K Amicon Ultra-15 columns (3000g, 15min at +25°C) and then washed/buffer exchanged 3 times with 15 ml of TEN 140 buffer with 0.1% Triton X100 (3000g, 5-15min at +25°C) in a fresh Amicon column. The resulting 0.5 ml concentrate was diluted with 2500 ul of RIPA buffer and transferred into two Eppendorf tubes. Biotin-Streptavidin affinity purification 150-300 ul of Streptavidin Agarose beads (Thermo Scientific, cat # 20349) were added to each tube. The mixture was rotated end-over-end for 12-16 hours at +16°C. Beads from both tubes were pooled into one 15 ml Falcon tube washed once with RIPA buffer, once with TEN 140 buffer with 0.1% Triton X100, twice with IgG Elution buffer and twice with IgG Elution buffer without SDS. For each washing step, beads were mixed with 12 ml of a given buffer on a rotating wheel for 10 min at +25°C and spun down for 5 min at 1000 g, +25°C. Beads were re-suspended in 10 ml of TEN 140 buffer and divided into three tubes: 6 ml for protein work, 2 ml for DNA and 2 ml for RNA analysis.
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 12 cycles and library fragments from 300-700 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq 2000 following the manufacturer's

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Data processing

Illumina base-calling pipeline; Heclios base-calling pipeline
for Illumina data: aligned to dm3 using bowtie (-t 1 -k 1); Helicos data was aligned using helisphere pipeline with default parameters, and uniquely aligned reads only.
conservative estimates of enrichment calculated using SPP (http://compbio.med.harvard.edu/Supplements/ChIP-seq/), using window size of 5Kb.
Genome_build: hg18
Supplementary_files_format_and_content: TDF files (for IGV browser) containing conservative estimates of smoothed enrichment

Submission date

Dec 19, 2013

Last update date

May 15, 2019

Contact name

Peter Kharchenko

Organization name

Harvard Medical School

Department

DBMI

Lab

Kharchenko