 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Aug 28, 2013 |
| Title |
seq-SDQ4154_IMB1_N2_Mxemb_Input_Rep2 |
| Sample type |
SRA |
| |
|
| Source name |
seq-SDQ4154_IMB1_N2_Mxemb_Input_Rep2
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
developmental stage: Mixed Embryo
genotype: wild type
sex type: mixed Male and Hermaphrodite population
|
| Growth protocol |
Worm_embryo_growth_and_harvest_v5. Embryos were prepared by bleaching from gravid N2 adults grown in standard S-basal media liquid culture. Live embryos were cross-linked in M9 + 2% formaldehyde for 30 minutes at room temperature followed by quenching with 125mM glycine for 5 minutes. Embryos were then washed twice with M9 Buffer and once by FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1 % sodium deoxycholate; 150 mM NaCl). Pellets were frozen at -80C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Worm_embryo_extraction_v5. Embryos were resuspended in FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1 % sodium deoxycholate; 150 mM NaCl) + protease inhibitors (Calbiochem Cat# 539131)+phosphatase inhibitors(Calbiochem cat#524624). Using a Branson sonifier microtip, samples were sonicated on ice at the following settings: 35% amplitude, 0.9 sec on, 0.1 sec off, 12 pulses, 10 times. Cell debris was removed by centrifuging at 13,000 g for 15 minutes at 4ºC and taking the supernatant. Protein concentration was determined by Bradford Assay and extracts were aliquoted at stored at -80C. For a detailed protocol see http://www.modencode.org/.
Worm_chromatin_immunoprecipitation_v5. 2mg extract + 1%sarkosyl was used for each ChIP with 10% taken as input directly into elution buffer (1% SDS in TE, 250 mM NaCl) and treated with RNAse. Antibody was added to each IP sample and incubated overnight at 4C. Immune complexes were incubated (2hrs at 4C) with 50 ul of IgG dynabeads (Dyna), and washed 5 minutes with 1.5mL of each of the following solutions: ChIP Buffer, ChIP Buffer+1M NaCl, ChIP Buffer+500mM NaCl, TEL Buffer (10mM Tris-HCl pH 8.0, 250mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1mM EDTA), and 2X TE (10mM Tris-HCl pH 8.0, 1mM EDTA). Samples were eluted twice with 150uL elution buffer for 15minutes at 65C. Samples were treated with proteinase K at 55C for 1-2hrs then transfer to 65C overnight to reverse crosslinks. DNA was cleaned up Qiagen PCR purification kit and concentrated using by Speed vac.
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_over_reps: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 |
SAMN02338625 |
| SRA |
SRX495046 |
| Named Annotation |
GSM1217466_KI026_control_afterfiting_all.wig.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1217466_KI026_control_afterfiting_all.wig.gz |
26.4 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 |
|
|
|
|
 |
