 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Aug 28, 2013 |
| Title |
seq-SDQ3948_COH3_FEM2_AD_Input_Rep2 |
| Sample type |
SRA |
| |
|
| Source name |
seq-SDQ3948_COH3_FEM2_AD_Input_Rep2
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: fem-2(b245)
developmental stage: Germline containing young adult
genotype: fem-2(b245)III
Sex: Hermaphrodite
|
| Growth protocol |
Worm_adult_growth_and_harvest_vHP2. Synchronized cultures of worms were grown until gravid in 400 mL batches. Gravid adults were isolated through sucrose floatation, and frozen into pellets in liquid nitrogen with EBS (250 mM HEPES (pH 7.5), 1180 mM NaCl, 480 mM KCl, 20 mM EDTA, 5 mM EGTA, sucrose (final 340 mM) and protease/phosphatase inhibitors. The pellets were stored at -80°C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Worms are frozen, ground, crosslinked for 20 minutes in EGS, and for an additional 10 minutes in EGS plus formaldehyde. Later, washed pellets are resuspended in FA buffer and subjected to sonication in Bioruptor (1 hour 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 extract from adult worms and 5 ?g of affinity purified antibody was used for ChIP. Dynal Protein A or G beads were used to recover the ChIPed DNA. Samples were then treated with RNase A and Proteinase K, and reverse crosslinked at 65°C. ChIPed DNA was purified and then quantified using the Pico Green HS fluorescent assay.
DNA for Library Prep is incubated with an End Repair Enzyme mix (NEB Klenow, T4 DNA polymerase and T4 PNK) to ensure blunt ends. It is then purified and incubated with Exo(-) Klenow fragment in the presence of dATP to add adenosine at the 3? ends (a single A-overhang for more efficient and directed ligation of the adaptors). After a second purification, the DNA fragments is ligated with single-end ?Homebrew? adaptors which contain an index sequence within (Corbin Jones? lab). After ligation, the samples are purified TWICE using SPRI beads, allowing for a size selection step getting rid of excess adaptors. Samples are then amplified by PCR with single end primers. Depending on the original fragmentation of the DNA, samples can either be purified by SPRI or by separation and purification from an agarose gel.
Prepared sample are sequenced using Illumina GAII or HiSeq2000 at the High Throughput Sequencing Facility of University of North Carolina at Chapel Hill or Cambridge.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
input DNA
|
| Data processing |
ChIP-seq_alignment-BWA:JL:1 protocol. BWA is a fast light-weighted tool that aligns relatively short sequences to a sequence database. The algorithms is based on Burrows-Wheeler Transform (BWT). This protocal, was ran as DEFAULT for both bwa aln and bwa samse.bwa aln: Find the SA coordinates of the input reads. Maximum maxSeedDiff differences are allowed in the first seedLen subsequence and maximum maxDiff differences are allowed in the whole sequence.OPTIONS:-n NUM
Maximum edit distance if the value is INT, or the fraction of missing alignments given 2% uniform base error rate if FLOAT. In the latter case, the maximum edit distance is automatically chosen for different read lengths. [0.04]-o INT
Maximum number of gap opens [1]-e INT
Maximum number of gap extensions, -1 for k-difference mode (disallowing long gaps) [-1]-d INT
Disallow a long deletion within INT bp towards the 3?-end [16]-i INT
Disallow an indel within INT bp towards the ends [5]-l INT
Take the first INT subsequence as seed. If INT is larger than the query sequence, seeding will be disabled. For long reads, this option is typically ranged from 25 to 35 for ?-k 2?. [inf]-k INT
Maximum edit distance in the seed [2]-t INT
Number of threads (multi-threading mode) [1]-M INT
Mismatch penalty. BWA will not search for suboptimal hits with a score lower than (bestScore-misMsc). [3]-O INT
Gap open penalty [11]-E INT
Gap extension penalty [4]-R INT
Proceed with suboptimal alignments if there are no more than INT equally best hits. This option only affects paired-end mapping. Increasing this threshold helps to improve the pairing accuracy at the cost of speed, especially for short reads (~32bp).-c
Reverse query but not complement it, which is required for alignment in the color space.-N
Disable iterative search. All hits with no more than maxDiff differences will be found. This mode is much slower than the default.-q INT
Parameter for read trimming. BWA trims a read down to argmax_x{sum_{i=x+1}^l(INT-q_i)} if q_l<INT where l is the original read length. [0] bwa samse:Generate alignments in the SAM format given single-end reads. Repetitive hits will be randomly chosen.OPTIONS:-n INT
Maximum number of alignments to output in the XA tag for reads paired properly. If a read has more than INT hits, the XA tag will not be written. [3] Processed data are obtained using following parameters: genome version is ce10 ChIP-Seq_MACS_Peak-calling:JL:1 protocol. Peak calls were determined by using Model-based Analysis of ChIP-Seq (MACS) software. MACS was supplied with the following parameters:tagSize = 25mfold = 10,30genomeSize = 90000000bandwidth = 300pvalue = 1e-5 Processed data are obtained using following parameters: genome version is ce10
|
| |
|
| Submission date |
Aug 27, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
DCC modENCODE |
| E-mail(s) |
help@modencode.org
|
| Phone |
416-673-8579
|
| Organization name |
Ontario Institute for Cancer Research
|
| Lab |
modENCODE DCC
|
| Street address |
MaRS Centre, South Tower, 101 College Street, Suite 800
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 0A3 |
| Country |
Canada |
| |
|
| Platform ID |
GPL9309 |
| Series (1) |
|
| Relations |
| BioSample |
SAMN02338454 |
| SRA |
SRX494891 |
| Named Annotation |
GSM1217308_SDQ3948_COH-3_csr7_control_afterfiting_all.wig.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1217308_SDQ3948_COH-3_csr7_control_afterfiting_all.wig.gz |
26.7 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
