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| Status |
Public on Aug 28, 2013 |
| Title |
seq-SDQ3528_NURF1_N2_L3_Input_Rep1 |
| Sample type |
SRA |
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| Source name |
seq-SDQ3528_NURF1_N2_L3_Input_Rep1
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
developmental stage: L3 Larva
genotype: wild type
sex type: mixed Male and Hermaphrodite population
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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 |
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 (20-30 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 ?l 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 Illuimina TruSeq DNA Sample Prep Kit.
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.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
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| Description |
input DNA
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| 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 wiggles are ce6, peak calls are 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 wiggles are ce6, peak calls are ce10
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| Submission date |
Aug 27, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
DCC modENCODE |
| E-mail(s) |
help@modencode.org
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| Phone |
416-673-8579
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| Organization name |
Ontario Institute for Cancer Research
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| Lab |
modENCODE DCC
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| Street address |
MaRS Centre, South Tower, 101 College Street, Suite 800
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| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 0A3 |
| Country |
Canada |
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| Platform ID |
GPL9309 |
| Series (1) |
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| Relations |
| BioSample |
SAMN02338719 |
| SRA |
SRX494970 |
| Named Annotation |
GSM1217389_Input_NA_mE16_E_N2_L3_aligned_linear_10bp_AA004_Pf203860.wig.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1217389_Input_NA_mE16_E_N2_L3_aligned_linear_10bp_AA004_Pf203860.wig.gz |
28.7 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 |
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