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Sample GSM6936031

Query DataSets for GSM6936031

Status

Public on Aug 21, 2023

Title

DMR, Heart, input, DMR11, vl0053

Sample type

SRA

Source name

Heart

Organism

Fukomys damarensis

Characteristics

tissue: Heart
genotype: wild-type
developmental stage: adult
age (months): 48 (estimated)
Sex: male
individual: DMR11
antibody: input

Treatment protocol

We performed chromatin immunoprecipitation experiments followed by high throughput sequencing (ChIP-seq) or 4C-seq using liver and heart tissue samples isolated from naked mole-rat, Damaraland mole-rat, guinea pig and mouse. At least two independent biological replicates from different animals were performed for each species and antibody. Wherever possible, tissues from young adult males were used. Tissues were prepared immediately post-mortem (typically within an hour) to maximize experimental quality. Post-mortem tissues were kept on ice until processed to minimize potential loss of protein-DNA interactions during post-mortem time. Sample allocations to experimental batches were randomised to ensure unbiased distributions of species, tissue, individual and sex, using the R/Bioconductor package OSAT (YAN et al. 2012).

Extracted molecule

genomic DNA

Extraction protocol

Tissues were prepared by direct perfusion of the liver with PBS, followed by dicing the whole organs (liver and heart) in small pieces around 1cm3. Blood clots within the heart ventricles were removed. Cross-linking of the diced tissue was performed in 1% formaldehyde solution for 20 min, addition of 250 mM glycine and incubation for a further 10 min to neutralize the formaldehyde. After homogenization of cross-linked tissues in a dounce tissue grinder, samples were washed twice with PBS and lysed according to published protocols (SCHMIDT et al. 2009 for ChIP and Krijger et al. 2020 for 4C) to solubilize DNA-protein complexes. For ChIP, chromatin was fragmented to 300 bp average size by sonication on a Misonix sonicator 3000 with a 418 tip (1/16 inch diameter). Chromatin from 50-200 mg of dounced tissue was used for each ChIP experiment using antibodies against H3K4me3 (millipore 05-1339), H3K27ac (abcam ab4729), H3K4me1 (abcam ab8895), TBX5 (Insight bio sc-515536), RXRA (sc-553), FOXA1 (ab70382), HNF4A (ARP31946), CEBPA (sc-9314) and HNF6 (sc-13050). For 4C, locus-specific 4C templates were made using two rounds of enzyme digestion (primary and secondary) and ligation. Purified 4C templates were amplified by inverse PCR with bait-specific primers.
Illumina sequencing libraries were prepared from ChIP-enriched or 4C-enriched DNA using ThruPLEX DNA-seq library preparation kit (Takara Bio) or NEBNext Ultra II DNA Library Prep Kit for Illumina with up to 10ng of input DNA and 8-15 PCR cycles. After PCR, libraries were pooled in equimolar concentrations and sequenced on Illumina HiSeq 4000, NextSeq 500 or NovaSeq 6000 instruments.
ChIP-Seq, 4C-Seq

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NextSeq 500

Data processing

ChIP-seq peak files were generated my MACS2 callPeak function (default parameters).
4C data was processed with pipe4C (Krijger et al. 2020)
Supplementary files format and content: MACS2 peak files for each ChIP and input combination (.narrowPeak extension)
Supplementary files format and content: 4C wig files for each 4C experiment

Submission date

Jan 16, 2023

Last update date

Aug 21, 2023

Contact name

Diego Villar

E-mail(s)

[email protected]

Phone

+447775847664

Organization name

Queen Mary University of London