Antigen

Antigen Class

TFs and others

Antigen

Tcf3

Cell type

Cell type Class

Neural

Cell type

Neural Stem Cells

MeSH Description

Self-renewing cells that generate the main phenotypes of the nervous system in both the embryo and adult. Neural stem cells are precursors to both NEURONS and NEUROGLIA.

Sample

source_name

neuronal stem cells

strain

C57BL/6J

genotype

wildtype

age

E12.5 embryo

fixation

20' 1.5 mM EGS, 10' 1% FA

antibody

E2A (sc-349X, Santa Cruz Biotechnology)

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Embryonic NSCs (approx. 10 x 10^6 cells) were fixed with 1.5 mM ethylene glycol bis[succinimidyl succinate] in DMSO for 20 min at RT, followed by adding formaldehyde to a final concentration of 1%, incubated for 10 min at RT and finally quenched by 0.2 M glycine. Cells were then lysed and sonicated with a microtip for 10 seconds followed by a 1 min break for 10 cycles. After adding 1% Triton-X to the sonicated lysates, ChIP was performed overnight at 4°C with Dynabeads M-280 Sheep anti-Rabbit IgG (Thermo Fisher, 11203D) linked to rabbit anti-E2A (1.5 µg, sc-349X, Santa Cruz Biotechnology). 10% of sonicated suspension was kept as input control. After washing and elution of chromatin-antibody complexes from the beads at 65°C overnight, crosslinking was reversed by proteinase K and RNase digestion. Genomic DNA and DNA from the sonicated input were purified using a PCR Purification Kit (Qiagen). Library preperation was performed from 2 ng of ChIP DNA using the Kapa Hyper Prep Kit with PCR library amplification (#KK8504, KapaBiosystems) according to the manufactur's protocol. Shortly, ChIP DNA was end-repaired and A-tailed before adapter ligation. After adapter ligation, the ChIP DNA was purified with Agencourt AMPure XP beads (#A63880, Beckman Coulter) and size-selected for 360-600 bp using the Pippin Prep System from Saga Science. The size-selected library is amplified by PCR using the Kapa Hyper Prep Kit (#KK8504,KapaBiosystems) and purified terminally with Agencourt AMPure XP beads (#A63880, Beckman Coulter).

Sequencing Platform

instrument_model

Illumina HiSeq 3000

mm10

Number of total reads

44320853

Reads aligned (%)

92.9

Duplicates removed (%)

73.2

Number of peaks

3185 (qval < 1E-05)

mm9

Number of total reads

44320853

Reads aligned (%)

92.8

Duplicates removed (%)

73.1

Number of peaks

3158 (qval < 1E-05)