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Sample GSM3121108

Query DataSets for GSM3121108

Status

Public on Aug 22, 2018

Title

N2 Input rep2

Sample type

SRA

Source name

embryonic lysate

Organism

Caenorhabditis elegans

Characteristics

antibody: none
genotype: N2 wild-type Bristol

Growth protocol

Mix-staged embryos were collected from adult worms by bleaching. Embryos were shaken at 200rpm at 20oC in CSM medium (100mM NaCl, 5.6mM K2HPO4, 4.4mM KH2PO4, 10μg/ml cholesterol, 10mM potassium citrate, 2mM CaCl2, 2mM MgSO4, 1X trace metals.) without food. Once the embryos hatched and became L1s, concentrated NA22 bacteria were added to the culture. Synchronized embryos were harvested by bleaching after 62-66 hours when most worms carried 1-8 embryos, frozen in liquid nitrogen and stored at -80oC.

Extracted molecule

genomic DNA

Extraction protocol

H3K9me2 ChIP was done as described previously (Hsu et. al, Science 2015) with the following changes: Embryos frozen in liquid nitrogen were thawed on ice and fixed in 1.5% Formaldehyde (Electron Microscopy Sciences, #15686) for 15 minutes at room temperature. Using the QSonica Q800 Sonicator, samples were sonicated at 30% amplitude, 30 seconds ON, 30 seconds OFF for a total of 15 minutes at 4oC, yielding 100-300 base pair fragments. 6μl of MABI0307 H3K9me2 antibody was coupled to 25μl beads (Pierce Protein A/G magnetic beads, #88802) for 8 hours at 4oC prior to ChIP. 40 μg of chromatin was used per ChIP reaction and chromatin was pre-cleared for 2 hours at 4°C using uncoupled magnetic beads (Pierce #88802). To elute the bound immunocomplexes, 150 μL of elution buffer (50mM NaHCO3, 140mM NaCl, 1% SDS) was added to each tube and heated at 65°C for 15 minutes.
The ChIP-Seq libraries were generated by using the Apollo 324 System and PrepX ILM DNA Library Kit from IntergenX. After adaptor ligation, the input and ChIP DNA were enriched by PCR amplification using NEBNext High-Fidelity 2X PCR master Mix with Q5 polymerase and PrepX PCR primer with the following PCR conditions: 30 seconds at 98°C, [10 seconds at 98°C, 30 seconds at 60°C, 30 seconds at 72°C] for 8 cycles for Input libraries and 11 cycles for ChIP libraries, followed by 5 minutes at 72°C (~15 uL adaptor ligated DNA, 25 uL NEBNext High-Fidelity 2X PCR master Mix, 2uL Universal PCR primer, brought to to 50ul with Nuclease-Free water). The enriched DNA was then purified using 50 uL (1:1 ratio of DNA volume and beads) of PCR Clean DX Beads (Aline) and size selected by Pippin Prep, 180-600bp. 1 uL of each library was applied to measure the concentration using a Qubit dsDNA assay kit (Invitrogen). 1 ng of DNA from each library was checked by a TapeStation (Agilent Technologies). Input and ChIP libraries were pooled such that they had the same expected number of sequence reads.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Data processing

Reads were aligned to the C. elegans reference genome (ce10) with Bowtie2 (version 2.3.1), using default parameters and '--very-sensitive'.
Log-likelihood-ratio tracks were created using MACS2 (version 2.1.1.20160309). For each sample: $ macs2 callpeak -t ${sample}_ChIP.bam -c ${sample}_Input.bam -f BAMPE -g ce --keep-dup all --bdg --SPMR; macs2 bdgcmp -t ${sample}_treat_pileup.bdg -c ${sample}_control_lambda.bdg -o ${sample}_logLR.bdg -m logLR -p 0.00001
LogLR tracks were converted to BigWig using linux sort and the UCSC tool bedGraphToBigWig. For each sample: $ sort -k1,1 -k2,2n ${sample}_logLR.bdg > ${sample}_logLR_sort.bdg; bedGraphToBigWig ${sample}_logLR_sort.bdg ce10genome.txt ${sample}_logLR.bw
Correlations of the logLR-bigWig files were calculated using the UCSC tool wigCorrelate.
Genome_build: ce10
Supplementary_files_format_and_content: bigWigs of log-likelihood-ratio tracks produced by 'macs2 bdgcmp'

Submission date

Apr 30, 2018

Last update date

Jul 10, 2024

Contact name

Susan E. Mango

E-mail(s)

[email protected]

Organization name

Harvard University

Department

Molecular and Cellular Biology