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| Status |
Public on Jun 01, 2018 |
| Title |
Capped nuclear RNA-seq of wild-type L1 larvae, replicate 1 |
| Sample type |
SRA |
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| Source name |
L1 larvae
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: wild-type N2
molecule: chromatin associated RNA
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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. glp-1(e2144) were raised at 15°C on standard NGM plates seeded with OP50 bacteria. Embryos were obtained by bleaching gravid adults and then approximately 6000 placed at 25°C on 150mm 2% NGM plates seeded with a 30X concentrated overnight culture of OP50. For harvest, worms were washed 3X in M9 and then worm slurry was frozen into popcorn by dripping into liquid nitrogen and stored at -80°C. Harvest times after embryo plating were D1/YA (53 hrs), D2 (71 hrs), D6 (167 hrs), D9 (239 hrs), D13 (335 hrs).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Nuclei were isolated and then chromatin associated RNA (development series) or nuclear RNA (ageing series) was isolated. Chromatin associated RNA was isolated as in (Pandya-Jones and Black 2009), resuspending washed nuclei in Trizol for RNA extraction. To isolate nuclear RNA, nuclei were directly mixed with Trizol. Following purification, RNA >200nt was isolated using Zymo clean and concentrate columns.
RNA was treated with DNase I, then with 5' Terminator nuclease (Epibio) to remove rRNA, and then stranded libraries prepared using the NEB Next Ultra Directional RNA Library Prep Kit (#E7420S). Libraries were made from two biological replicates for each developmental stage and each ageing time point.
RNA-seq of >200nt capped nuclear 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 to 20bp, and aligned using bwa in paired-end mode.
Low-quality (q<10), mitochondrial, rRNA, and modENCODE-blacklisted reads were discarded.
Strand-specific paired-end coverage with regions between read pairs "filled in" were generated.
Read coverage was normalised by DESeq2 sizeFactors of gene-level read counts.
Stage-specific normalised coverage was calculated by averaging across two biological replicates, and smoothed by binning at 10bp resolution.
Normalised coverage was log2-scaled using the formula log2(n + 1).
Genome_build: WBcel215/ce10 (WS220)
Supplementary_files_format_and_content: Forward and reverse-strand normalised genome-wide read coverage in linear and log2 scales.
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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) |
| GSE114483 |
Chromatin accessibility dynamics across C. elegans development and ageing [lcap] |
| GSE114494 |
Chromatin accessibility dynamics across C. elegans development and ageing |
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| Relations |
| BioSample |
SAMN09210909 |
| SRA |
SRX4085158 |
| 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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