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| Status |
Public on Apr 02, 2015 |
| Title |
Input DNA for ChIPs, lin-35 mutant extract IL3 |
| Sample type |
SRA |
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| Source name |
L3 larvae
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| Organism |
Caenorhabditis elegans |
| Characteristics |
tissue: whole larvae
strain: JA1507
Stage: L3
genotype/variation: lin-35 mutant
chip antibody: none
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| Treatment protocol |
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.
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| Growth protocol |
Worm_L3_growth_and_harvest_vPK1. About 2-7 million of 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 50ul of worms are stained for DAPI 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 (exceptional antibodies due to better results are incubated 2hrs). 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. DNA was incubated with an enzyme mix (Klenow, T4 DNA polymerase and T4 PNK) to ensure blunt ends and then with Exo(-) Klenow fragment in the presence of dATP to add adenosine at the 3' ends. The DNA fragments were ligated with appropriate adaptor (Illumina) and then amplified by PCR. The amplicon was loaded into an agarose gel, and size-selected DNA was recovered from the gel. Prepared sample are sequenced using Illumina GAIIx or HiSeq2000 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 Genome Analyzer IIx |
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| Description |
Sample name: input_lin35_IL051_IL3
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| Data processing |
ChIP-seq and RNA-seq reads were aligned to the WS220 assembly of the C. elegans genome using BWA (Li and Durbin, 2010) with default settings.
ChIP-seq data were normalized using BEADS algorithm (Cheung et al, 2011).
ChIP-seq signals were exported to bigWig tracks at native resolution.
RNA-seq tag counts for each gene were extracted from BAM alignment files using HTSeq method working in ”union” mode. An exon model based on WS235 genes (taken from Ensembl Gene 74 December 2013) was lifted over to ce10/WS220.
RNA-seq tag counts were used to build the expression matrix. Differential gene expression between N2, lin-35 mutant, and htz-1 mutant was tested using DESeq2
Genome_build: ce10/WS220
Genome_build: http://ws220.wormbase.org/
Genome_build: http://hgdownload.soe.ucsc.edu/goldenPath/ce10/bigZips/
Supplementary_files_format_and_content: "linear" scores in bigWig tracks represent BEADS values; files were generated using BEADS normalization pipeline in R
Supplementary_files_format_and_content: "zscore" scores in bigWig tracks represent standardized BEADS values; files were generated using BEADS normalization pipeline in R
Supplementary_files_format_and_content: xslx spreadsheet contains: reads per kilobase (kb) of transcript per millions reads in library (RPKM) for all RNA-seq experiments, averaged RPKM values for replicates, plus log2 fold changes, p-values and adjusted p-values (FDR) calculated with DEseq2 for gene knock-out replicates versus N2 (wild-type) replicate
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| Submission date |
Oct 29, 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 |
GPL13776 |
| Series (1) |
| GSE62833 |
The DREAM complex promotes gene body deposition of H2A.Z for target repression |
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| Relations |
| BioSample |
SAMN03152262 |
| SRA |
SRX747307 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1534082_Input_NA_il3_F_lin35_L3_aligned_linear_1bp_IL051_Pea07217.bw |
177.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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