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| Status |
Public on Jun 01, 2018 |
| Title |
wt_L1_ATAC-seq_rep1 |
| Sample type |
SRA |
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| Source name |
L1 larvae
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| Organism |
Caenorhabditis elegans |
| Characteristics |
age: L1 larvae
strain: wild-type N2
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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 |
genomic DNA |
| Extraction protocol |
Frozen embryos or worms were broken by grinding in a mortar and pestle or smashing using a Biopulverizer, then the frozen powder was thawed in 10 ml Egg buffer (25 mM HEPES pH 7.3, 118 mM NaCl, 48 mM KCl, 2 mM CaCl2, 2 mM MgCl2). Ground worms were pelleted by spinning at 1500 g for 2 minutes, then resuspended in 10ml working Buffer A (0.3M sucrose, 10 mM Tris pH 7.5, 10 mM MgCl2, 1mM DTT, 0.5 mM spermidine 0.15 mM spermine, protease inhibitors (Roche complete, EDTA free) containing 0.025% IGEPAL CA-630. The sample was dounced 10X in a 14ml stainless steel tissue grinder (VWR), then the sample spun 100g for 6 min to pellet large fragments. The supernatant was kept and the pellet resuspended in a further 10 ml Buffer A, then dounced for 25 strokes. This was spun 100g for 6 min to pellet debris and the supernatants, which contain the nuclei, were pooled, spun again at 100g for 6 min to pellet debris, and transferred to a new tube. Nuclei were counted using a hemocytometer. Twenty million nuclei were transferred to a 1.5 ml tube and spun 2000g for 10min to pellet.
ATAC-seq was performed essentially as in (Buenrostro et al. 2013). Twenty million nuclei were resuspended in 47.5 ul of tagmentation buffer, incubated for 30 minutes at 37°C with 2.5 ul Tn5 enzyme (Illumina Nextera kit), and then tagmented DNA purified using a MinElute column (Qiagen) and converted into a library using the Nextera kit protocol. Typically, libraries were amplified using 12–16 PCR cycles.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| 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, and modENCODE-blacklisted reads were discarded.
Normalised coverage was calculated using MACS2: --format BAM --bdg --SPMR --gsize ce --nolambda --nomodel --extsize 150 --shift -75 --keep-dup all
Stage-specific accessibility was calculated by averaging across biological replicates, and smoothed by binning the signal at 10bp resolution.
Genome_build: WBcel215/ce10 (WS220)
Supplementary_files_format_and_content: Stage-specific genome-wide accessibility profiles. Normalized data across replicates.
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| Submission date |
May 14, 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) |
| GSE114439 |
Chromatin accessibility dynamics across C. elegans development and ageing [ATAC-seq] |
| GSE114494 |
Chromatin accessibility dynamics across C. elegans development and ageing |
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| Relations |
| BioSample |
SAMN09208013 |
| SRA |
SRX4082384 |
| 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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