 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 01, 2018 |
| Title |
wt_L3_DNase-seq_rep1_800U_ml |
| Sample type |
SRA |
| |
|
| Source name |
L3 larvae
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: wild-type N2
age: L3 larvae
protocol: 1
|
| 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).
|
| 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.
Replicate concentration courses of DNase I were performed for each stage as follows. Twenty million nuclei were digested for 10 minutes at 25C using 2.5, 5, 10, 25, 50, 100, 200, and 800 units/ml DNase I (Roche), then EDTA was added to stop the reactions. Embryo micrococcal nuclease (MNase) digestion concentration courses for embryos were made by digesting nuclei with 0.025, 0.05, 0.1, 0.25, 0.5, 1, 4, 8, or 16 units/ml MNase in 10mM Tris pH 7.5, 10mM MgCl2, 4mM CaCl2 for 10 minutes at 37C. Reactions were stopped by the additon of EDTA. Following digestions, total DNA was isolated from the nuclei following proteinase K and RNase A digestion, then large fragments removed by binding to Agencourt AMPure XP beads (0.5 volumes). Small double cut fragments < 300 bp were isolated either using a Pippen prep gel (protocol 1) or using Agencourt AMPure XP beads (protocol 2). DNA was converted into sequencing libraries using the Illumina Truseq kit.
|
| |
|
| Library strategy |
DNase-Hypersensitivity |
| Library source |
genomic |
| Library selection |
DNAse |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Reads were trimmed using trim_galore, and aligned using bwa in paired-end mode.
Improperly paired, low-quality (q<10), mitochondrial, and modENCODE-blacklisted reads were discarded.
Normalised coverage was calculated from <300bp reads using MACS2: --format BAMPE --bdg --SPMR --gsize ce --nolambda, and smoothed by binning the signal at 10bp resolution.
Genome_build: WBcel215/ce10 (WS220)
Supplementary_files_format_and_content: Genome-wide accessibility profiles by stage/concentration/replicate.
|
| |
|
| Submission date |
May 15, 2018 |
| Last update date |
Jun 01, 2018 |
| Contact name |
Julie Ahringer |
| E-mail(s) |
ja219@cam.ac.uk
|
| Organization name |
University of Cambridge
|
| Department |
The Gurdon Institute
|
| Street address |
Tennis Court Road
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 1QN |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL13657 |
| Series (2) |
| GSE114481 |
Chromatin accessibility dynamics across C. elegans development and ageing [DNase, MNase] |
| GSE114494 |
Chromatin accessibility dynamics across C. elegans development and ageing |
|
| Relations |
| BioSample |
SAMN09211019 |
| SRA |
SRX4085399 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3142700_dnase_wt_l3_rep1_800U_ml.bw |
57.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
