Antigen

Antigen Class

Input control

Antigen

Input control

Cell type

Cell type Class

Pluripotent stem cell

Cell type

Embryoid Bodies

MeSH Description

Spontaneous aggregations of human embryonic stem cells that occur in vitro after culturing in a medium that lacks LEUKEMIC INHIBITORY FACTOR. The embryoid bodies can further differentiate into cells that represent different lineages.

Sample

source_name

ES-derived embryoid bodies with induced transcription factors

strain

Ainv15(iAscl1[bHLH:Ngn2].HA)

chip target

input

chip antibody

none

treatment

Dox induction + 12 hours

cell type

Embryoid Bodies

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

ChIP-seq: Cells were collected at 12 hours and 48 hours after TF induction and fixed with 1mM DSG (ProtoChem) followed by 1% FA (vol/vol) each for 15 min at room temperature. Pellets containing 25-30x10^6 cells were aliquoted and flash-frozen at -80°C. Cell aliquots were thawed on ice and resuspended in 5 ml of cold lysis buffer 1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol (vol/vol), 0.5% Igepal (vol/vol), 0.25% Triton X-100 (vol/vol)) with 1X protease inhibitors (Roche, 11697498001) and incubated at 4°C on a rotator. The samples were centrifuged for 5 min at 2300 rpm at 4°C and resuspended in 5ml of cold lysis buffer 2 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol (vol/vol), 0.5% Igepal (vol/vol), 0.25% Triton X-100 (vol/vol)) with 1X protease inhibitors and incubated 10 min at 4°C on a rotator. Nuclear extracts were centrifuged for 5 min at 2300 rpm resuspended in cold 3 ml of sonication buffer (50mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate (wt/vol), 0.1% SDS (wt/vol). Sonication of the nuclear extracts was performed on ice with Branson 450 digital sonifier (Marshall Scientific, B450CC) at 20% amplitude, 18 cycles of 30s ON/60s OFF, to sheer crosslinked chromatin into average size of approximately 300 bp. Immunoprecipitation of the sonicated chromatin with Dynabeads protein-G (Thermo Fisher) conjugated antibodies were done overnight (16 hr) at 4°C on a rotator. 5 ug of following antibodies were used for immunoprecipitation. In-house library preparation kit was used for ChIP-seq: 24ul of ChIP DNA (1:100 dilution of input DNA) was used to prepare lllumina DNA sequencing libraries. Bioo Scientific multiplexed adapters were ligated after end repair and A-tailing, and unligated adapters were removed by purification using Agencourt AmpureXP beads (Beckman Coulter). Adapter-ligated DNA was amplified by PCR using TruSeq primers (Sigma). DNA libraries between 300 and 500 bp in size were purified from agarose gel purified using Qiagen minElute columns. and the final quantification of the library before pooling was done with KAPA library amplification kit (Roche Lightcycler 480).

Sequencing Platform

instrument_model

NextSeq 500

mm10

Number of total reads

33191686

Reads aligned (%)

95.4

Duplicates removed (%)

21.9

Number of peaks

41240 (qval < 1E-05)

mm9

Number of total reads

33191686

Reads aligned (%)

95.3

Duplicates removed (%)

22.0

Number of peaks

41163 (qval < 1E-05)