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| Status |
Public on Jun 01, 2018 |
| Title |
wt_L2_short_cap_RNAseq_rep2 |
| Sample type |
SRA |
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| Source name |
L2 larvae
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: wild-type N2
age: L2 larvae
protocol: 2
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| Growth protocol |
Wild-type N2 were grown at 20°C in liquid culture to the adult stage using standard S-basal medium with HB101 bacteria, animals bleached to obtain embryos, and the embryos hatched without food in M9 buffer for 24 hrs at 20°C to obtain synchronized starved L1 larvae. L1 larvae were grown in a further liquid culture at 20°C to the desired stage, then collected, washed in M9, floated on sucrose, washed again in M9, then frozen into "popcorn" by dripping embryo or worm slurry into liquid nitrogen. Popcorn were stored at -80°C until use. Times of growth were L1 (4 hrs), L2 (20 hrs), L3 (30 hrs), L4 (45 hrs), young adults (60 hrs). Mixed populations of embryos were collected by bleaching cultures of synchronized one day old adults.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Nuclei were isolated and then chromatin associated RNA was isolated as in (Pandya-Jones and Black 2009), resuspending washed nuclei in Trizol for RNA extraction. Following purification, RNA was of 17–200nt was isoated using Zymo clean and concentrate columns.
To profile transcription initiation ("short cap RNA-seq"), stranded libraries were prepared from 17–200nt RNA. Non-capped RNA was degraded by first converting uncapped RNAs into 5’-monophosphorylated RNAs using RNA polyphosphatase (Epibio), then treated with 5' Terminator nuclease (Epibio). The RNA was then treated with calf intestinal phosphatase to remove 5’ phosphates from undegraded RNA, and decapped using Tobacco Acid Pyrophosphatase (Epicentre, protocol 1), Cap-Clip Acid Pyrophosphatase (CellScript, protocol 2) or Decapping Pyrophosphohydrolase, (Dpph tebu-bio protocol 3) and then converted into sequencing libraries using the Illumina TruSeq Small RNA Preparation Kit kit. Libraries were size selected to be 145–225 bp long on a 6% acrylamide gel, giving inserts of 20–100 bp long. Libraries were made from two biological replicates for each developmental stage. During the course of this work, the TAP enzyme stopped being available; the Cap-Clip and Dpph enzymes perform less well than TAP. One L3 replicate and one YA replicate were made using a slightly different protocol (protocol 4) from Chen et al 2013 (doi: 10.1101/gr.153668.112).
RNA-seq of short (<100nt) capped chromatin associated RNA
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
CAGE |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
Reads were trimmed using trim_galore, and aligned using bwa in single-end mode.
Low-quality (q<10), mitochondrial, tRNA, snoRNA, rRNA, or miRNA, and modENCODE-blacklisted reads were discarded.
Strand-specific coverage of 5’ ends of reads was generated.
Stage-specific tracks: coverage of all 5' end reads in all replicates per stage.
Tracks pooled across all stages: coverage of 5' end reads was summed across all replicates in all stages, and filtered for reproducibility by only keeping signal at base pairs with non-zero coverage in at least two replicates.
Genome_build: WBcel215/ce10 (WS220)
Supplementary_files_format_and_content: Profiles of transcription initiation for each stage/strand; profiles of transcription initiation pooled across all stages & filtered for reproducible signal. For embyro (and pooled) data, we also included short cap replicates from (Chen et al., 2013; GSE42819).
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| Submission date |
May 15, 2018 |
| Last update date |
Jun 01, 2018 |
| Contact name |
Julie Ahringer |
| E-mail(s) |
ja219@cam.ac.uk
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| Organization name |
University of Cambridge
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| Department |
The Gurdon Institute
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| Street address |
Tennis Court Road
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| City |
Cambridge |
| ZIP/Postal code |
CB2 1QN |
| Country |
United Kingdom |
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| Platform ID |
GPL18730 |
| Series (2) |
| GSE114490 |
Chromatin accessibility dynamics across C. elegans development and ageing [scap] |
| GSE114494 |
Chromatin accessibility dynamics across C. elegans development and ageing |
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| Relations |
| BioSample |
SAMN09211072 |
| SRA |
SRX4085620 |
| 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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