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| Status |
Public on Apr 10, 2014 |
| Title |
H4K16ac ChIP-seq, rep2 |
| Sample type |
SRA |
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| Source name |
L3 larvae, H4K16ac ChIP
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain/background: N2
genotype/variation: wild-type
tissue: whole body
developmental stage: L3
Sex: mixed male and hermaphrodite
chip antibody: anti-H4K16ac
chip antibody supplier: Millipore
chip antibody catalog#: 07-329
chip antibody lot#: JBC1888188
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| Growth protocol |
Worm_L3_growth_and_harvest_vPK1. About 2-7 million worms are bleached and then hatched in M9 for 24-42 hrs. About 100 embryos are seeded onto the plate to test for contamination and hatching efficiency. Remaining hatched L1 larvae are inoculated in a proper volume of liquid culture. Next day when larvae reach the L3 stage, they are cleaned by M9 washes and sucrose gradient and collected by freezing in liquid nitrogen. Just before collection, DIC pictures are taken and about 100 worms are assayed by DAPI staining to assess the stage.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Worm_L3_extraction_vPK1. Worms are frozen, ground, and crosslinked for 10 minutes in 1% formaldehyde. Formaldehyde is quenched and cross-linked tissue washed, then resuspended in FA buffer and subjected to sonication in Bioruptor (14 pulses of 30 seconds with 1 minute rests in between). Extracts are then spun down and soluble fraction is stored for quality tests and future ChIP.
Worm_chromatin_immunoprecipitation_vIL2. Appropriate amount of extract is incubated overnight with a proper amount of antibody (normally 500ug-1mg extract with 2-3ug antibody). Afterwards, 40ul of equilibrated magnetic beads (either protein A or G, depending on antibody) are added and incubated for 2 hrs. Later, washes with FA, 500mM-salt FA, 1M salt FA, TEL, and TE buffer are performed and DNA is eluted in elution buffer (1% SDS in TE with 250 mM NaCl) two times with 57 ul volume each, at 65°C. Samples are treated with RNase, proteinase K and then crosslinks are reversed overnight at 65°C. DNA is purified on Qiagen PCR purification columns, tested by q-PCR for ChIP quality, and stored at -20°C for future applications.
ChIP or input DNA is blunt ended, A-tailed, ligated to adaptors, amplified by PCR, then size selected using AMPure beads. The protocol uses the Illumina TruSeq DNA Sample Prep Kit. Prepared samples are sequenced using Illumina GAII or HiSeq 2000 at the High-Throughput Sequencing Facility of University of North Carolina at Chapel Hill or Cambridge.
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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 |
ChIP-seq reads were aligned to the WS220 assembly of the C. elegans genome using BWA (Li and Durbin, 2010) with default settings.
Data were normalized using BEADS algorithm (Cheung et al., 2011).
Signals were exported to bigWig tracks at 1bp resolution.
Genome_build: WS220
Supplementary_files_format_and_content: Normalized bigWig track files were generated using BEADS pipeline in R; Scores represent BEADS normalized values (linear fold enrichment relative to input).
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| Submission date |
Apr 08, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Przemyslaw Aleksander Stempor |
| Organization name |
University of Cambridge
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| Department |
The Gurdon Institute
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| Lab |
Ahringer Lab
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| Street address |
Tennis Court Road
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| City |
Cambridge |
| State/province |
United Kingdom |
| ZIP/Postal code |
CB2 1QN |
| Country |
United Kingdom |
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| Platform ID |
GPL13657 |
| Series (1) |
| GSE56627 |
seq-UP07329_H4K16ac:JBC1888188_N2_L3 |
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| Relations |
| BioSample |
SAMN02724794 |
| SRA |
SRX514568 |
| Named Annotation |
GSM1366018_H4K16ac_UP07329_fE6_F_N2_L3_NORM_linear_1bp_AA190_F7208412.bw |
| Named Annotation |
GSM1366018_H4K16ac_UP07329_fE6_F_N2_L3_aligned_linear_1bp_AA190_Pcc08406.bw |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1366018_H4K16ac_UP07329_fE6_F_N2_L3_NORM_linear_1bp_AA190_F7208412.bw |
78.2 Mb |
(ftp)(http) |
BW |
| GSM1366018_H4K16ac_UP07329_fE6_F_N2_L3_aligned_linear_1bp_AA190_Pcc08406.bw |
36.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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