Antigen

Antigen Class

TFs and others

Antigen

Ets1

Cell type

Cell type Class

Blood

Cell type

Thymocytes

MeSH Description

HEMATOPOIETIC PROGENITOR CELLS that have migrated to the THYMUS where they differentiate into T-LYMPHOCYTES. Thymocytes are classified into maturational stages based on the expression of CELL SURFACE ANTIGENS.

Sample

source_name

primary Rag2 -/- thymocytes

strain

C57BL/6

age

5-6 weeks

genotype/variation

Rag2 -/-

cells used

1.0E+08

antibody

Santa Cruz (sc-350x)

antibody quantity

20 µg

magnetic beads

Sheep anti-rabbit IgG dynabeads

bead volume

200µl

estimated fragment size

136bp

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Phenol-chroloform For ChIP-Seq, thymocytes were aliquoted in crosslinked pellets of 5 107 cells and lysed by rotation for 10 min at 4°C in 2x 1.25ml LB1 (50mM Hepes pH 7.5, 140mM NaCl, 1mM EDTA, pH 8, 10% glycerol, 0.75% NP-40, 0.25% Triton X-100). Following centrifugation at 1,350g for 5 minutes at 4°C, nuclei were subsequently treated with 2x1.25ml LB2 (200mM NaCl, 1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8) on a rotating wheel for 10 minutes at 4°C. Chromatin was resuspended in 1.5ml LB3 (1mM EDTA pH8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 100mM, NaCl, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine) in 15ml polypropylene falcon tubes and then sonicated for 10 cycles (30 seconds on, 30 seconds off) at 40W using a VCX130 sonicator (VibraCell) down to an average size of about 250bp. 50µl aliquots were collected as Input controls. All buffers contained final concentrations of 1X protease inhibitors (Roche, France) as well as 0.2mM PMSF and 1μg /ml pepstatin. ChIPs were conducted using either α-rabbit (Ets1) or protein-G (all other ChIPs) DynaBeads (Invitrogen).Antibody and bead mixes were washed three times with 1ml BSA blocking buffer (0.5% BSA in 1x DPBS), incubated and rotated overnight at 4°C, and subsequently washed another three times with 1ml BSA blocking buffer. Immunoprecipitation was carried out overnight in 750µl LB3 at 4°C on a rotating wheel. Samples were then washed 8 times in 1ml RIPA buffer (50mM Hepes pH 7.6, 500mM LiCl, 1mM EDTA pH 8, 1% NP-40, 0.7% Na-Deoxycholate) and once in 1ml TE buffer with 50mM NaCL. Two 15-min and 10-min subsequent elutions were carried out in a waterbath at 65°C, vortexing every other minute, for a final volume of 100µl. 100µl 1X elution buffer (50mM Tris pH 8, 10mM EDTA pH 8, 1% SDS) was added to eluates which were reverse-crosslinked overnight in a 65°C waterbath. Equal volumes of TE buffer (100µl) were added to samples as well as 0.2μg/ml RNAse A and placed at 37°C for 2 hours, followed by proteinase K treatment at 0.2μg/ml at 55°C. DNA purification was carried out via phenol-chloroform extraction followed by elution using QiaQuick PCR purification kits. (QiaGen) Samples were pooled per replicate then SpeedVac-ed down to 30µl. DNA quantification of eluates was obtained using Bioanalyzer High Sensitivity chips (Agilent). Library preparation was carried out as per manufacturer’s instructions (Illumina) using 10ng material. Samples were subsequently size-selected via gel migration and extraction, with average cut sizes at about 230 bp for DN and 250 bp for DP, including adapters. Sequencing was performed using either Genome Analyzer II (Illumina) or SOLiD 5500xl sequencers (Applied Biosystems), whereby samples were sequenced at the CNAG, Barcelona, Spain and TAGC, Marseille, France, respectively. For SOLiD 5500xl sequencing, ChIP-seq libraries were been prepared according to the Life Technologies protocol, SOLiD ChIP-seq kit guide (A12066), using 15 PCR cycles. The quality of the libraries was checked with the BioAnalyzer DNA High sensitivity and by Q-PCR.

Sequencing Platform

instrument_model

Illumina Genome Analyzer II

mm10

Number of total reads

33922533

Reads aligned (%)

94.6

Duplicates removed (%)

15.3

Number of peaks

1011 (qval < 1E-05)

mm9

Number of total reads

33922533

Reads aligned (%)

94.4

Duplicates removed (%)

15.4

Number of peaks

1119 (qval < 1E-05)