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| Status |
Public on Feb 21, 2019 |
| Title |
glp-4 input rep3 |
| Sample type |
SRA |
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| Source name |
whole worm
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: SS104 glp-4(bn2) I.
Stage: L4
chip antibody: MRG-1 antibody (Novus bio)
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| Treatment protocol |
For experiments, worms were synchronized by bleaching and L1 larvae were put on OP50 containing plates and grown at 25°C until most of the animals reached L4 stage.
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| Growth protocol |
C. elegans strains were maintained on OP50 bacteria and NGM agar plates at 15ºC. Worms were transferred to fresh food once a week.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Synchronized L4s were washed with M9 buffer and flash-frozen as “worm popcorn” in liquid nitrogen. The popcorn was pulverized using a biopulverizer before further grinding to a fine powder using a mortar. The powder was dissolved in 10 vol 1,1 % formaldehyde in PBS+1 mM PMSF and fixed for 10 min with gentle rocking. Quenching was achieved by adding 2,5 M Glycine to a final concentration of 125 mM and gently rocking for 5 min. After centrifugation the pellet was washed with ice-cold PBS+1 mM PMSF, before it was resuspended in FA-buffer (50 mM HEPES/KOH pH 7,5, 1mM EDTA, 1% Triton X-100, 0,1 % sodium deoxycholate, 150 mM NaCl) + 1 % Sarkosyl + protease inhibitor and sonicated twice using a Bioruptor (15 times, 15 sec ON, 15 sec OFF; high settings) followed by 15 min centrifugation at full speed, 4°C. The supernatant was taken off (approx. 2-4 mg protein) and incubated either with MRG-1 antibody (Novus) or with buffer ON at 4°C on a rotator. Next, samples were incubated with µMACS ProteinA beads (Milteny Biotec) for 1 h on ice before they were applied to µMACS magnetic M columns that were equilibrated using FA buffer. The columns with bound material were washed 2x using FA buffer followed by washing with FA buffer + 1 mM NaCl and FA buffer + 500 mM NaCl. After further washing with TEL buffer (0,25 mM LiCl, 1 % sodium deoxycholate, 1 mM EDTA) and 2x with TE buffer, the samples were eluted using elution buffer (1 % SDS, 250 mM NaCl, 10 mM Tris pH 8,0, 1mM EDTA). The fixation was reverse crosslinked using 2 µl of 10 mg/ml Proteinase K at 50°C for 1 h followed by incubation at 65°C ON. The DNA was purified using the QIAquick PCR purification kit in a final volume of 40 µl.
Libraries were prepared using the NEXTflex qCHIP-Seq v2 kit (NOVA-5130-12) according to manufacturer’s instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Basecalling and Demultiplexing was done with Illumina bcl2fastq v2.18.0.12 software.
ChIP-seq reads were mapped using bowtie2 v2.3.2 in paired end mode with default settings (-D 15 -R 2 -N 0 -L 22 -i S,1,1.15) and allowing up to one alignment per read (-k 1 ) to version ce10 of the worm genome assembly.
Peaks were called for each replicate using the MACS v2.1.0 module callpeak with those settings: ( -g ce -keep-dup auto -q 0.05 - nomodel -extsize 300).
Genome_build: ce10
Supplementary_files_format_and_content: Bigwig files were generated by converting bam files to bed files using bedtools bamtobed v2.25.0 , followed by an extension of each alignment region by 200 bases up and down-stream, respectively. Regions overlapping chromosome borders were clipped. Then base resolution coverage score was calculated and scaled to reads-per-million by dividing each position by one million. Finally the scaled coverage track was exported to a bigwig file . NarrowPeak files were generated during the peak calling process and the format follows the ENCODE narrowPeak format specifications.
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| Submission date |
Feb 21, 2018 |
| Last update date |
Feb 21, 2019 |
| Contact name |
Baris Tursun |
| Organization name |
Max Delbrück Center for Molecular Medicine
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| Street address |
Robert-Rössle-Str. 10
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| City |
Berlin |
| ZIP/Postal code |
13092 |
| Country |
Germany |
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| Platform ID |
GPL22765 |
| Series (1) |
| GSE110969 |
Automated RNAi screening reveals MRG-1/MRG15 as a barrier for germline to neuron reprogramming in C. elegans |
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| Relations |
| BioSample |
SAMN08579944 |
| SRA |
SRX3733156 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3019578_glp4_no.AB3.bw |
139.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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