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Sample GSM4173749

Query DataSets for GSM4173749

Status

Public on Nov 19, 2019

Title

Lonp1_IP

Sample type

SRA

Source name

whole worm

Organism

Caenorhabditis elegans

Characteristics

strain background: N2
genotype/variation: LONP1-FLAG
developmental stage: L4 stage hermaphrodite worms
chip antibody: Monoclonal ANTI-FLAG M2 antibody (Sigma, F1804)

Treatment protocol

The worms were lysed via Teflon homogenizer in cold PBS with protease inhibitors (Roche). Cross-linking of DNA and protein was performed by treating the worms with 1.85% formaldehyde for 15 min. Glycine was added to a final concentration of 125mM for 5 min at room temperature to quench the formaldehyde. The pellets were resuspended twice in cold PBS with protease inhibitor. Samples were sonicated in Bioruptor (Diagenode) for 15 min at 4°C on high intensity (30s on and 30s off). Samples were transferred to microfuge tubes and spun at 15,000*g for 15 min at 4°C. The supernatant was precleaned with pre-blocked ChIP-grade Pierce™ magnetic protein A/G (Thermo Scientific) and then incubated with Monoclonal ANTI-FLAG® M2 antibody (Sigma, F1804) or Mouse mAb IgG1 Isotype Control (Cell Signaling Technology, G3A1) rotating overnight at 4°C. Then, the antibody-chromatin complex was precipitated with magnetic beads or protein A sepharose beads (Invitrogen) dependent on the host of generated antibodies. After washing, the crosslinks were reversed by incubation at 65 °C overnight and further treated with RNaseA 37 °C for 1 hour and then proteinase K 55 °C for 2 hours, respectively. Finally, immunoprecipitated and input DNA were purified with ChIP DNA Clean & Concentrator (Zymo Research, D5205) and used as templates for qPCR or next generation sequencing.

Growth protocol

Synchronized worms were cultured in liquid and harvested at early L4 stage by sucrose flotation.

Extracted molecule

genomic DNA

Extraction protocol

Synchronized worms were cultured in liquid and harvested at early L4 stage by sucrose flotation. The worms were lysed via Teflon homogenizer in cold PBS with protease inhibitors (Roche). Cross-linking of DNA and protein was performed by treating the worms with 1.85% formaldehyde for 15 min. Glycine was added to a final concentration of 125mM for 5 min at room temperature to quench the formaldehyde. The pellets were resuspended twice in cold PBS with protease inhibitor. Samples were sonicated in Bioruptor (Diagenode) for 15 min at 4°C on high intensity (30s on and 30s off). Samples were transferred to microfuge tubes and spun at 15,000*g for 15 min at 4°C. The supernatant was precleaned with pre-blocked ChIP-grade Pierce™ magnetic protein A/G (Thermo Scientific) and then incubated with Monoclonal ANTI-FLAG® M2 antibody (Sigma, F1804) or Mouse mAb IgG1 Isotype Control (Cell Signaling Technology, G3A1) rotating overnight at 4°C. Then, the antibody-chromatin complex was precipitated with magnetic beads or protein A sepharose beads (Invitrogen) dependent on the host of generated antibodies. After washing, the crosslinks were reversed by incubation at 65 °C overnight and further treated with RNaseA 37 °C for 1 hour and then proteinase K 55 °C for 2 hours, respectively.
Finally, immunoprecipitated and input DNA were purified with ChIP DNA Clean & Concentrator (Zymo Research, D5205) and used as templates for qPCR or next generation sequencing.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina MiSeq

Data processing

Basecalls performed using CASAVA version 1.8
he quality of the raw sequencing data was first evaluated with fastqc (0.11.5). ChIP-seq reads were aligned to the WBcel220(ce10) worm genome by using by Burrows-Wheeler Aligner (BWA MEM, BWA version 0.7.15) algorithm with the standard default settings.
The duplicated reads were removed using picard tools v1.96.
Peaks were determined using MACS version 2.135 with the no-model parameter.IgG was used as a control for peak-calling, and the bigwig files were generated with the signal as fold enrichment by macs2 following the procedure: ChIP threshold (0.2), Enrichment Fold (2.5), Rescue Fold (3).
Genome_build: WBcel220(ce10)
Supplementary_files_format_and_content: ChIP-seq bigwig files were generated using MCAS 2.135; Scores represent the ChIP-seq tag numbers,

Submission date

Nov 18, 2019

Last update date

Nov 20, 2019

Contact name

pengpeng liu

E-mail(s)

[email protected]

Organization name

umassmed

Department

MCCB