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| Status |
Public on Jan 01, 2017 |
| Title |
pre-mRNA-seq_met-2set-25_OP50 |
| Sample type |
SRA |
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| Source name |
Young adult whole animal
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| Organism |
Caenorhabditis elegans |
| Characteristics |
genotype/variation: met-2(n4256);set-25(ok5021)
temperature: 19˚C
feeding: OP50
antibody: Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2)
molecule subtype: RNA extracted from anty-Pol II (S2) IP fragmented chromatin
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| Growth protocol |
C. elegans strains N2, hrde-1 (tm1200), set-32(ok1457), met-2(n4256);set-25(ok5021), and met-2(n4256);set-25(ok5021);set-32(ok1457) were cultured on NGM plates with E. coli OP50 as the food source in a temperature controlled incubator at 19°C. Synchronized worms were raised at 19°C and young adult worm samples were collected.
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| Extracted molecule |
total RNA |
| Extraction protocol |
100-200 μl frozen synchronized young adult worm pellets were used for each pre-mRNA-seq experiment. Crushed pellets (pulverized by grinding in liquid nitrogen with a mortar and pestle) were resuspended in 1ml of pre-chilled RIPA buffer (1X PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1X HALT combined protease and phosphatase inhibitor cocktail [ThermoScientific]). To crosslink, formaldehyde was added to the crude extract to a final concentration of 2%. The lysate was rotated at 4°C for 10 minutes. 0.1 ml of 1M Tris-HCl (pH 7.5) was added to quench formaldehyde. Lysate was spun at 6000 x g for 30 second and the supernatant was removed. The pellet was resuspended in 0.5ml of pre-chilled RIPA buffer. The lysate was transferred to a 1.5 ml TPX tube (Diagenode) and sonicated (Bioruptor [Diagenode], output level: high, interval: 0.5, three times of 8-minute sonication). For each immumoprecipiation experiment, 2 μg of anti-Pol II-S2 (ab5095, Abcam) was added to 400 μl of the lysate (containing approximately 50 μg DNA). The IP mix was rotated overnight at 4°C. 50 μl of Protein A Dynabeads (Life Technology) was added and rotated for another 2 hours. The beads were then washed three times with 800 μl ice-cold LiCl washing buffer (100 mM Tris-HCl, pH7.5, 500 mM LiCl, 1% NP-40 and 1% sodim deoxycholate). To elute the immunoprecipitation product and reverse crosslink, beads were incubated with 400 μl of worm lysis buffer (0.1 M Tris-HCl, pH 7.5, 100 mM NaCl, 50 mM EDTA, 1% SDS, and 200 μg/ml of proteinase K) at 65 °C for 1.5-2 hours with agitation every 30 minutes and then subject to organic extraction and precipitation of total DNA and RNA. DNA/RNA mix was treated with DNase I (NEB) and resulted RNA was used for the pre-mRNA-seq library preparation.
pre-mRNA library was prepaed using the following protocol: RNA was fragmented by hydrolysis using Fragmentation Reagent Kit (Ambion). RNA was treated with T4 PNK (NEB). Then RNA 3’ end was ligated to IDT linker-1 (5’-rAppCTGTAGGCACCATCAATC-3’) using T4 RNA ligase 2, truncated (NEB, M0242S). RNA library of about 35-60 nt was size selected using 8 % polyacrylamide (acrylamide:bis-acrylamide = 19:1) gels containing 8 M urea (size selection also eliminated excess IDT linker-1). RNA 5’end was ligated to DNA-RNA hybrid oligo (5’-ACGCTCTTCCGATCTrNrNrNrN-3’, where rNrNrNrN is a 4-nt barcode) using T4 RNA ligase 1 (NEB) with ATP. First strand cDNA was synthesized using SuperScript III RT kit (Life Technologies) and primer SG-460 (5’-GGAGTTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG-3’). cDNA library was PCR amplified at early cycle (tested by different cycle number to capture linear amplification phase and avoid over amplification). PCR was done using a common primer SG-465 (5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’) and primer with 6-nt index for multiplexing (5’-CAAGCAGAAGACGGCATACGAGAT[ index]GTGACTGGAGTTCAGACGTGTGCTCTTCC-3’). Amplified library was size selected by 2% agarose gel and gel purified using QIAquick Gel Extraction Kit (QIAGEN).
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
50 nt Illumina sequencing reads were aligned to the C. elegans genome using bowtie[0.12.7].
rpkm value for each 1kb is calculated by the number of reads that perfectly aligned to both strands of 1kb-non-overlaping regions, normalized by the sequencing depth of the library in million.
Genome_build: WS190/ce6
Supplementary_files_format_and_content: .xlsx file containing rpkm values of all 1kb regions for 5 pre-mRNA-seq libraries. GRH and GRTS regions are previously defined germline nuclear RNAi-dependent heterochromatin (GRH) and transcriptional silencing (GRTS) targets.H3K9me3 ChIP-seq results of OP50 feeding experiments were used as replica 1 to identify different HMT-dependent K9 targets replica 1. H3K9me3 ChIP-seq results from RNAi control experiment (GFP RNAi) were used as replica 2 to identify HMT-dependent K9 targets replica 2. All replica HMT-dependent K9 target lists are reported in the table. In addition, miss-annotated regions are also reported in the table. Whole genome classification of H3K9me3 level is also labeled in the table as K9 tire: 1-3 correspond to the top 5% H3K9me3 regions, 4 correspond to the next 5-25%, tire 5 is 25-50%, tire 6 is 50-75%, tire 7 is 75-100% (bottom H3K9me3 quartile, correspond to the background level) excluding tire 8 - extreme low values (=0 or ~0).
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| Submission date |
Sep 07, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Sam Guoping Gu |
| E-mail(s) |
ggu@dls.rutgers.edu
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| Organization name |
Rutgers University
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| Department |
Molecular Biology and Biochemistry
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| Street address |
604 Allison Road
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| City |
Piscataway |
| State/province |
New Jersey |
| ZIP/Postal code |
08854 |
| Country |
USA |
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| Platform ID |
GPL13657 |
| Series (2) |
| GSE86516 |
Decoupling the downstream effects of germline nuclear RNAi reveals that transcriptional repression and heritable RNAi are independent of the H3K9me3 response in C. elegans [pre-mRNA-seq] |
| GSE86517 |
Decoupling the downstream effects of germline nuclear RNAi reveals that transcriptional repression and heritable RNAi are independent of the H3K9me3 response in C. elegans |
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| Relations |
| BioSample |
SAMN05736294 |
| SRA |
SRX2144202 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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