 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 21, 2019 |
| Title |
H3K27me3 lin37 26C rep1 |
| Sample type |
SRA |
| |
|
| Source name |
H3K27me3 lin37 26C
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
developmental stage: L1
genotype: lin-37(n758)
temperature: 26C
chip antibody: Fujifilm Wako MABI0323, #309–95259
input control: Input lin37 26C rep1
extract_protocol: Method 2
|
| Treatment protocol |
Frozen late L1s were ground using a mortar and pestle with liquid nitrogen. The resulting worm powder was resuspended in FA buffer and crosslinked for 10 minutes in 1% formaldehyde.
|
| Growth protocol |
Synchronized N2 or mutant L1s from standard NGM plates were washed off of plates and grown in standard S-basal media liquid culture with HB101 as a food source at 20°C until there were gravid adults. Adult worms were treated with standard bleaching solution to collect embryos, and embryos were incubated for 14 hours in S-basal without food to acquire synchronized L1s. For 20°C samples F1 synchronized N2 or mutant L1s were grown in standard S-basal media liquid culture with HB101 as a food source at 20°C until there were gravid adult, they were bleached, and the resulting embryos were grown for 14 hours until all L1s then frozen in liquid nitrogen. For 26°C samples F1 synchronized N2 or mutant L1s were grown in standard S-basal media liquid culture with HB101 as a food source at 20°C until the L4 larval stage at which point the culture was shifted to 26°C and grown until there were gravid adults. The adults were bleached and the resulting embryos were grown for 14 hours at 26°C until all L1s then frozen in liquid nitrogen
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Crosslinked samples were resuspended in FA buffer, and sonicated in a Diagenode Bioruptor (60 pulses of 30 seconds at full power with 1 minute rest in between). Extracts were clarified and protein concentrations determined using a Bradford reagents. Histone modifications were individually IPed using 1 μg appropriate antibodies on prepared lysates. ChIPs were performed with 0.5 mg of extract, and 2% of the extract was set aside for an input reference control. Two methods were used for ChIP, see Sample section above to match with sample. Method 1: ChIPs were incubated overnight at 4°C with 1% sarkosyl. Mouse IgG Dynabeads equilibrated in 100 μL FA buffer were added and incubated for 2 hours at 4°C. ChIPs were washed with the following buffers: twice with FA buffer, once with FA buffer containing 1 M NaCl, once with FA buffer containing 0.5 M NaCl, once with TEL buffer (10 mM Tris-HCl pH 8.0, 0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA), and twice with TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA). 2 elutions of 150 μL elution buffer containing TE plus 1% SDS and 250 mM NaCl were incubated at 65°C. Eluted ChIP and input samples were incubated with RNAse One for 30 min at 37°C and then proteinase K for 1 hour at 55°C. Crosslinks were reversed overnight at 65°C. DNA was purified using a Qiagen PCR clean-up column and eluted in 25 μl of water. Method 2: ChIPs were performed using an IP-Star Compact Automated System (Diagenode) according to the manufacturers’ instructions with the following settings and reagents: the “indirect method” was used with 100 μL reaction, 80 μL Mouse IgG Dynabeads equilibrated in FA buffer; IP for 12 hr, bead incubation for 2 hr; 10 minute washes at middle speed with FA buffer containing 1 M NaCl, FA buffer containing 0.5M NaCl, TEL buffer, and TE buffer. Samples were eluted in 100 μL Elution Buffer. Eluted samples were incubated with RNAse One for 30 min at 37°C and then proteinase K for 1 hour at 55°C. Crosslinks were reversed overnight at 65°C. DNA was purified by phenol chloroform extraction, ETOH precipitated, and DNA resuspended in 20 μl of water.
Two methods were used for library construction, see Characteristics section. Method 1: Both ChIP and input DNA samples had the ends blunted and phosphorylated with KlenowDNAP, T4 DNAP, and T4 PNK. 3’-dA overhangs are then added with Klenow Fragment (3’ to 5’exo minus), and Illumina adapters were subsequently ligated to the ends. Next, PCR amplification was performed using Illumina primers, for a total of 16 cycles. Amplified DNA libraries were size-selected on a 2% agarose gel so that fragments sized between 250-350bp were obtained; gel extraction was carried out at room temperature. Method 2: The NEBNext Ultra DNA library Prep Kit (NEB) was used following the manufacturers’ instructions. 1 ng of starting DNA was used, adapters were diluted 1:40, and AMPure beads were used for size selection before amplification to enrich for fragments corresponding to a 200 bp insert size.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Raw sequence reads from the Illumina HiSeq (50 bp single-end read sequencing for ChIP-seq) were mapped to the C. elegans genome (Wormbase version WS220) using Bowtie 1.1.2 with default settings.
MACS2 was used to call peaks and to create bedgraph files. The broad peak option was used for H3K9me2. The statistical cutoff q=0.01 was used. Further options used were -g ce --bdg —keep-dup=auto.
Custom scripts were used to scale bedgraph files to the same number of total reads (number depended on the histone mark).
Genome_build: ws220 (ce10)
Supplementary_files_format_and_content: bedgraph file for genome browser containing scaled read coverage
Supplementary_files_format_and_content: narrowPeak or broadPeak file with peak calls as determined by MACS2
|
| |
|
| Submission date |
Feb 21, 2019 |
| Last update date |
Feb 22, 2019 |
| Contact name |
Lisa N. Petrella |
| E-mail(s) |
lisa.petrella@marquette.edu
|
| Organization name |
Marquette University
|
| Department |
Biological Sciences
|
| Street address |
1428 W. Clybourn St
|
| City |
Milwaukee |
| State/province |
WI |
| ZIP/Postal code |
53233 |
| Country |
USA |
| |
|
| Platform ID |
GPL22765 |
| Series (2) |
| GSE126871 |
Repression of germline genes in C. elegans somatic tissues by H3K9 dimethylation of their promoters [ChIP-seq] |
| GSE126884 |
Repression of germline genes in C. elegans somatic tissues by H3K9 dimethylation of their promoters |
|
| Relations |
| BioSample |
SAMN10984041 |
| SRA |
SRX5402704 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3615944_H3K27me3.lin37.26C.rep2.i15.SSJK001_broadq0.01.dupauto_treat_pileup_sc.15Mil.bedgraph.gz |
94.0 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
