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Sample GSM2495705

Query DataSets for GSM2495705

Status

Public on Oct 25, 2017

Title

LIN-35 ChIP-seq in lin-35(n745) late embryos, rep 2

Sample type

SRA

Source name

LIN-35 ChIP-seq in lin-35(n745) late embryos

Organism

Caenorhabditis elegans

Characteristics

genotype: lin-35(n745)
tissue: late embryo
chip-antibody: SDIX/Novus Biologicals Q2003
input control: lin-35(n745) extract rep 2

Treatment protocol

Frozen late embryos were ground using a mortar and pestle and crosslinked for 10 minutes in 1% formaldehyde.

Growth protocol

Synchronized N2 or lin-35(n745) L1s were grown in standard S-basal media liquid culture for 72 or 96 hours, respectively. Adult worms were treated with standard bleaching solution to collect embryos, and embryos were incubated for up to 3.5 hours 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, protein concentrations determined using a Qubit fluorometer, and precleared with Protein A Dynabeads before proceeding to ChIP. DRM subunits were individually IPed using 1-5 μg appropriate antibodies on prepared lysates. ChIPs were performed with 1-2 mg of extract, and 2% of the extract was set aside for an input reference control. ChIPs were incubated overnight at 4°C with 1% sarkosyl. Protein A Dynabeads equilibrated in 20 μL FA buffer were added and incubated for 2 hours at 4°C. ChIPs were washed with the following buffers: 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 50 μL elution buffer containing TE plus 1% SDS and 250 mM NaCl were incubated at 55°C. Eluted ChIP and input samples were incubated with proteinase K for 1 hour at 55°C. Crosslinks were reversed overnight at 65°C. DNA was purified by phenol-chloroform extraction and ethanol precipitation using glycogen as a carrier.
Libraries from wild-type late embryos, extracts rep 1a, 1b, 2a, and 2b: Both ChIP and input DNA samples have their 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 are subsequently ligated to the ends. Next, PCRamplification is performed using Illumina 1.1 and 2.1 primers, for a total of 18 cycles. AmplifiedDNA libraries are size-selected on a 2% agarose gel so that fragments sized between 250-350bp are obtained; gel extraction is carried out at room temperature. Libaries from wild-type late embryo extract rep 3 and lin-35(n745) late embryo extracts rep 1-3 were prepared using the TruSeq ChIP Sample Prep Kit (Illumina). Amplifed libraries were size selected to obtain 200-500 base pair fragments using Agencourt AMPure XP beads (Beckman Coulter).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Description

processed data file:
LIN-35.SDQ2003.lin-35.n745.rep0.vsInputRep0.SES.bw

Data processing

bowtie 1.1.2 was run with the -m 2 -S options to map the Illumina reads to the genome version ws220/ce10
SPP v1.10.1 peak caller (from phantompeakqualtools) was run on the bowtie output for each subunit for each replicate (rep 1-3), pseudoreplicates (rep1.pr1,rep1.pr2,etc), pooled replicates (rep 0), and pooled psuedoreplicates (rep0.pr1,rep0.pr2) against pooled input with the following parameters: -npeak=40000, -savn, -savp, -rf
IDR v2.0.2 was run on SPP peak outputs for each subunit on replicates (rep1v2, rep1v3, rep2v3) defining N1-2, N1-3, and N2-3 (threshold IDR < 0.01), selfPseudoReplicates (rep1.pr1vpr2) defining N1, N2, and N3 (threshold IDR < 0.02), and pooledPseudoReplicates (rep0.pr1vpr2) defining Np (threshold IDR < 0.005). N1-2, N1-3, and N2-3 were averaged to define Nt for each factor, which was selected as the final number of peaks passing the 1% IDR threshold. The Rescue Ratio (Np/Nt) and Self-consistency ratio (Max(N1,N2,N3)/Min(N1,N2,N3)) were calculated to check for low consistency between replicates (low consistency is defined as ratio >> 2).
The deepTools v2.0 tool bamCompare was used to normalize to input and create bigwig signal track files for visualization. Pooled replicates were normalized to pooled input using the Signal Extraction Scaling (SES) method using the following parameters: --scaleFactorsMethod SES--numberOfSamples=100000 --ratio ratio --binSize 10 --pseudocount=1 --extendReads x (x was calculated from SPP analysis for each factor)
Genome_build: ws220 (ce10)
Supplementary_files_format_and_content: bigwig signal track files of combined replicates (rep0) for each ChIP normalized to pooled input using the SES method (Diaz et al. 2012) and narrowPeak files containing peak regions for each ChIP (IDR < 0.01)

Submission date

Feb 19, 2017

Last update date

May 15, 2019

Contact name

Paul Goetsch

Organization name

UCSC

Department

MCD Biology

Lab

Strome lab