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| Status |
Public on Nov 04, 2013 |
| Title |
DZ038_TRA1_N2L2 |
| Sample type |
SRA |
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| Source name |
N2 strain, L2 stage
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
treatment: no treatment
chip antibody: anti-TRA-1 antibody UMN163
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| Treatment protocol |
Frozen animals were physically disrupted by grinding in liquid nitrogen in a mortar and pestle, fixed in 1%PFA in PBS for 15 min at RT and post-fixed with 50mM glycine for 5 min at RT. All subsequent steps were performed at 4°C. Material was pelleted, washed 3x in PBS with protease inhibitors, and resuspended in 1%SDS lysis buffer. Fixed chromatin was sheared with a Diagenode Bioruptor Standard, on the high setting for 7.5 min total on time (15 min of 30 sec on, 30 sec off cycles).
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| Growth protocol |
Gravid hermaphrodites were bleached and their eggs were collected and allowed to hatch overnight in M9. Synchronized animals were then grown in standard liquid culture conditions (wormbook.org) using HB101 bacteria as a food source, and staged by gonad migration and time. N2, AF16, and tra-1(e1834)/hT2[bli-4(e937) let-?(q782) qIs48] animals were grown at 20°C; spe-11(hc77) and glp-4(bn2) animals were grown at 25°C. tra-1(e1834) homozygotes were sorted from tra-1(e1834)/hT2[bli-4(e937) let-?(q782) qIs48] siblings using a COPAS BIOSORT SELECT (Union Biometrica, 350-5000-000) to collect GFPnegative animals. Two consecutive rounds of sorting were used to ensure a pure population of tra-1(e1834) homozygotes. Collected animals were separated from residual bacteria by sucrose flotation and washes in M9, and then frozen in liquid nitrogen.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Insoluble material was pelleted at 12000g at 4C for 10 min. 1mL of supernatant was added to 9mL of dilution buffer. 300uL of this was set aside as an “input” sample. 2ug of TRA-1 antibody UMN163 was added to the remaining sample and incubated in a 15mL conical with rotation at 4C o/n. Samples were spun at 15000g at 4C for 15 min and supernatant was decanted into a fresh 15mL conical tube. 50ul of blocked 50% PAS beads were added and incubated with rotation at 4C for 1 hr. PAS beads were pelleted at 1000g for 30 sec and supernatant was removed. Beads were then washed once in each of the following buffers; low salt buffer, high salt buffer, LiCl buffer, Morohashi RIPA, DOC lysis buffer, and TE. Beads were then transferred to a fresh tube and washed again with TE. Each wash consisted of adding 1mL of 4C buffer, inverting for 30 seconds, spinning at 1000g at 4C for 30 sec, and then discarding the supernatant. After the final wash, residual TE was removed with a small bore pipette tip. 130uL of elution buffer was added to each sample and incubated with rotation at RT for 15 min. Beads were spun down at 1000g for 30 sec and 100uL of supernatant was transferred into a new 1.5mL tube. An additional 100uL of elution buffer was added to each sample and incubated with rotation at RT for an additional 15 min. Again, beads were spun down at 1000g for 30 sec and another 100uL of supernatant was transferred into the same 1.5mL tube, making 200uL total volume per sample. At this point, 200uL of the “input” sample was processed in parallel to the IP samples for the rest of the protocol. 10uL of 4M NaCl was added to each sample and incubated o/n at 65C. Samples were then cooled to RT, and 1uL of 10 mg/mL RNAse A was added to each sample and incubated at 37C for 30 min. Then 4uL of .5M EDTA, 8uL Tris (pH=6.5), and 1uL of PCR grade proteinase K were added to each sample and incubated at 50C for 1 hr. Samples were then purified using Qiagen PCR purification columns and eluted in 50-100uL of Qiagen EB.
DNA were blunt-ended, A-overhanged, and then ligated with adaptors. DNA libraries were amplified by PCR with single-end primers. Amplicons in the size range between 200 and 500 bp were recovered from agarose gel.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
AGATGGTT
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| Data processing |
All C. elegans reads were subsequently trimmed to the first 27 bases and aligned to the ce10 reference genome using Bowtie, accepting only uniquely mapped reads with up to two mismatches. For C. briggsae, 48-base reads were aligned using the same Bowtie program settings to the cb4 reference genome.
Aligned reads were extended to 200 bases, and the number of reads overlapping with each base of the genome was counted using Bedtools (Quinlan & Hall, Bioinformatics, 2010).
The per-base read counts were then normalized to the genome-wide average in order to take the sequencing depth into account.
Genome_build: ce10
Genome_build: cb4
Supplementary_files_format_and_content: Processed data files are in bigwig format representing overlappaing read counts at each base in the genome.
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| Submission date |
Jul 16, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Kohta Ikegami |
| E-mail(s) |
kohta.ikegami@cchmc.org
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| Organization name |
Cincinnati Children's Hospital Medical Center
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| Department |
Division of Molecular Cardiovascular Biology
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| Lab |
Ikegami Lab
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| Street address |
240 Albert Sabin Way, Cincinnati, OH 45229
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| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45229 |
| Country |
USA |
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| Platform ID |
GPL13657 |
| Series (1) |
| GSE48917 |
Chromatin Immunoprecipitation of TRA-1 in C. elegans and C. briggsae |
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| Relations |
| BioSample |
SAMN02256371 |
| SRA |
SRX323677 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1186792_DZ038_TRA1_N2L2_tr27_ce10_BTm1x200n.bw |
44.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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