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| Status |
Public on Jul 03, 2014 |
| Title |
CG3680 r1 pull-down |
| Sample type |
SRA |
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| Source name |
S2 cell line
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell line background: S2
pull-down tag: CG3680 BioTAP
platform: Illumina
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| Treatment protocol |
Harvested S2 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 Drosophila S2 cells, embryos, 3rd instar larvae or adult flies 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 (for 3rd instar larval or adult fly chromatin, a motorized Teflon pestle was used). 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 (for 3rd instar larvae or adult flies motorized Teflon pestle had been used). 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 3.5 min, 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.
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| Growth protocol |
Drosophila S2 cells were grown in four 50 ml large T-flasks (225 cm2) in 10% FBS –supplemented Schneider’s Drosophila medium (Invitrogen Cat # 11720) to a density of ~5x106 cells/ml. 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.). To generate 100 g of 6-20 hr embryos, flies were grown at 25°C with 65% humidity using large embryo collection cages (Flystuff cat. N. 59-101) containing molasses plates with yeast paste. 100 g of 3rd instar larvae or adult flies were used. Larvae were separated from the food by washing in 20% sucrose (see Ashburner, 1989). Flies were collected 1-12 hours after eclosion.
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| 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 (for 50 ml of 3rd instar larvae or adult fly chromatin 1 ml beads was used). 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 Genome Analyzer following the manufacturer's
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| 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.
smoothed enrichment calculated using SPP (http://compbio.med.harvard.edu/Supplements/ChIP-seq/), get.smoothed.enrichment.mle().
Genome_build: dm3 for D.melanogaster
Supplementary_files_format_and_content: bedGraph files containing either smoothed enrichment MLE (*IP.bedGraph), or strand-specific smoothed read density (*pos.bedGraph; *neg.bedGraph)
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| Submission date |
Apr 04, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter Kharchenko |
| Organization name |
Harvard Medical School
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| Department |
DBMI
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| Lab |
Kharchenko
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| Street address |
10 Shattuck St.
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL13304 |
| Series (1) |
| GSE56101 |
Heterochromatin-associated interactions of Drosophila HP1 with dADD1, HIPP1, and repetitive RNAs |
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| Relations |
| BioSample |
SAMN02719523 |
| SRA |
SRX511131 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1363107_CG3680.r1.ip.bedGraph.gz |
57.0 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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