Antigen

Antigen Class

TFs and others

Antigen

Isl1

Cell type

Cell type Class

Cardiovascular

Cell type

Sinoatrial Node

MeSH Description

The small mass of modified cardiac muscle fibers located at the junction of the superior vena cava (VENA CAVA, SUPERIOR) and right atrium. Contraction impulses probably start in this node, spread over the atrium (HEART ATRIUM) and are then transmitted by the atrioventricular bundle (BUNDLE OF HIS) to the ventricle (HEART VENTRICLE).

Sample

source_name

Sinoatrial node (SAN) cells

strain background

ICR

genotype/variation

Hcn4-H2BGFP

developmental stage

postnatal day1-day3

cell type

Sinoatrial node (SAN) cells

chip antibody

ISL1

chip antibody vendor

DSHB

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

SAN cells were first crosslinked in 2mM disuccinimidyl glutarate (Pierce) in PBS for 30 min, then subsequently in 1% formaldehyde (Sigma) in PBS for 10 min all at room temperature. The reactions were quenched by adding glycine (Sigma) to a final concentration of 125mM. The cells were immediately centrifuged (5 min, 700x g, 4°C) and washed twice with ice-cold PBS. Cells were resuspended in swelling buffer (10 mM HEPES/KOH pH7.9, 85 mM KCl, 1 mM EDTA, 0.5% IGEPAL CA-630 (Sigma), 1x protease inhibitor cocktail (Roche), 1 mM PMSF) for 5 min. Cell pellets were spun down and resuspended in 1ml RIPA buffer (10 mM Tris/HCl pH7.6, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton X-100, 1x protease inhibitor cocktail (Roche), 1 mM PMSF). Chromatin was sheared to an average DNA size of 100-400 bp by administering 10 pulses of 30s duration at 12 W power output with 60s pause on wet ice using a Misonix 3000 sonicator. The lysate was cleared by centrifugation (5min, 16000 x g, 4°C). 1% of the supernatant was kept as ChIP input. Meanwhile Protein G Dynabeads were prepared with the ISL1 antibody (39.4D5, DSHB) by incubating Dynabeads Protein G and 5 μg specific antibody in 0.5% BSA/PBS for 1 hr at 4°C on rotator, then washed twice with 0.5% BSA/PBS and brought up to the original volume with 0.1% BSA/PBS. The protein-DNA complex of interest was immunoprecipitated by rotating the supernatant with 30μl Dynabeads Protein G-coated with specific antibody overnight at 4°C (Dynabeads from Invitrogen). Beads were washed with each buffer by rotating in 1 ml buffer at 4°C for 5 min: RIPA buffer (10 mM Tris/HCl pH7.6, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton X-100, 1x protease inhibitor cocktail (Roche), 1 mM PMSF), LiCl buffer (0.25 M LiCl, 1% NP40, 1% NaDOC), TE plus 0.2% Triton X-100 and TE plus 50mM NaCl. Immunoprecipitated chromatin was eluted twice with 100 μl elution buffer each (TE, 2% SDS) into fresh tubes for 30 min and 10 min, respectively, eluates were pooled, the Na+ concentration was adjusted to 300 mM with 5 M NaCl and crosslinks were reversed overnight at 65°C in a hybridization oven. The samples were sequentially incubated at 37°C for 1hr each with 0.33 mg/ml RNase A and 0.5 mg/ml proteinase K. The DNA was isolated using the ChIP DNA Clean & Concentrator (Zymo Research) according to the manufacturer’s instructions. Sequencing libraries were prepared from collected ISL1 ChIP and corresponding input DNA by blunting, A-tailing, adaptor ligation using NExtFlex barcoded adapters from Bioo Scientific. Libraries were PCR-amplified for 12-15 cycles, size selected for 225-375bp fragments by gel extraction and single-end sequenced on a Hi-Seq 2500 (Illumina) for 50 cycles.

Sequencing Platform

instrument_model

Illumina HiSeq 2500

mm10

Number of total reads

9159045

Reads aligned (%)

95.2

Duplicates removed (%)

16.1

Number of peaks

322 (qval < 1E-05)

mm9

Number of total reads

9159045

Reads aligned (%)

95.0

Duplicates removed (%)

16.4

Number of peaks

305 (qval < 1E-05)