Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
twi

Cell type

Cell type Class
Embryo
Cell type
Mesoderm
NA
NA

Attributes by original data submitter

Sample

Alias
E-MTAB-4585:Twist-H2B
Broker name
ArrayExpress
Description
Protocols: Wildtype Drosophila melanogaster (Twist-H2B) flies were grown in population cages at 25 degr. Staged 2 hr populations of embryos were collected and aged at 25 degr till the required stage of development. Two independent collections were performed for each timepoint. The collected embryos were dechorionated using 50% bleach and formaldehyde fixed in 10 ml cross-linking solution (50 mM Hepes, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 1.8 % formaldehyde, pH 8.0) and 30ml n-heptane on a shaker table at RT for 15 minutes. The reaction was terminated with 125 mM glycine, 0.1% Triton X-100 in PBS. After washing out the fix, the embryos were blotted dry and frozen in liquid nitrogen. A small number of embryos from each collections was set aside to stage each collection. Fixed mesodermal nuclei were isolated and used subsequently to perform chromatin immunoprecipitation as previously described by Bonn and colleagues (Cell type-specific chromatin immuno precipitation from multicellular complex samples using BiTS-ChIP, Nat Prot, 2012). Briefly, whole embryos were homogenized and pushed through the needles to extract nuclei. Nuclei were stained with alpha-SBP antibody generated in mouse that recognizes SBP-tagged histone H2B. Subsequently, nuclei were stained with secondary antibody alpha-mouse Alexa Fluor 488, which was used to separate mesodermal fixed nuclei using Fluorescence Activated Cell Sorting (FACS) with purity greater than 95%. Several sorts were pulled together to obtain sufficient amount of material. Chromatin was sheared to 200 bp with Bioruptor and used to perform imunoprecipitation (IP) as previously described in Sandmann et al. (Nat Prot, 2006) with antibodies (generous gifts from Joerg Mueller recognizing Pho (2-382 aa) on 4-6h and 6-8h, or dSfmbt (531-980 aa) on 6-8h material. The Extraction is part of IP described in the sample's ChIP protocol NGS libraries were prepared as previously described by Bonn and colleagues (Cell type-specific chromatin immuno precipitation from multicellular complex samples using BiTS-ChIP, Nat Prot, 2012).
ENA checklist
ERC000011
INSDC center name
EMBL
INSDC first public
2017-04-11T17:01:57Z
INSDC last update
2016-04-14T11:30:37Z
INSDC status
public
SRA accession
ERS1115623
Sample Name
ERS1115623
Title
Twist-H2B
age
6-8h
initial time point
egg laying
organism part
mesoderm
sex
mixed sex
strain
Twist-H2B

Sequenced DNA Library

library_name
Polycomb_Pho_6-8h_twi-ST-H2B_R1
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Wildtype Drosophila melanogaster (Twist-H2B) flies were grown in population cages at 25 degr. Staged 2 hr populations of embryos were collected and aged at 25 degr till the required stage of development. Two independent collections were performed for each timepoint. The collected embryos were dechorionated using 50% bleach and formaldehyde fixed in 10 ml cross-linking solution (50 mM Hepes, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 1.8 % formaldehyde, pH 8.0) and 30ml n-heptane on a shaker table at RT for 15 minutes. The reaction was terminated with 125 mM glycine, 0.1% Triton X-100 in PBS. After washing out the fix, the embryos were blotted dry and frozen in liquid nitrogen. A small number of embryos from each collections was set aside to stage each collection. Fixed mesodermal nuclei were isolated and used subsequently to perform chromatin immunoprecipitation as previously described by Bonn and colleagues (Cell type-specific chromatin immuno precipitation from multicellular complex samples using BiTS-ChIP, Nat Prot, 2012). Briefly, whole embryos were homogenized and pushed through the needles to extract nuclei. Nuclei were stained with alpha-SBP antibody generated in mouse that recognizes SBP-tagged histone H2B. Subsequently, nuclei were stained with secondary antibody alpha-mouse Alexa Fluor 488, which was used to separate mesodermal fixed nuclei using Fluorescence Activated Cell Sorting (FACS) with purity greater than 95%. Several sorts were pulled together to obtain sufficient amount of material. Chromatin was sheared to 200 bp with Bioruptor and used to perform imunoprecipitation (IP) as previously described in Sandmann et al. (Nat Prot, 2006) with antibodies (generous gifts from Joerg Mueller recognizing Pho (2-382 aa) on 4-6h and 6-8h, or dSfmbt (531-980 aa) on 6-8h material. The Extraction is part of IP described in the sample's ChIP protocol NGS libraries were prepared as previously described by Bonn and colleagues (Cell type-specific chromatin immuno precipitation from multicellular complex samples using BiTS-ChIP, Nat Prot, 2012).

Sequencing Platform

instrument_model
Illumina Genome Analyzer IIx

dm3

Number of total reads
23182678
Reads aligned (%)
25.2
Duplicates removed (%)
73.6
Number of peaks
10005 (qval < 1E-05)

dm6

Number of total reads
23182678
Reads aligned (%)
22.3
Duplicates removed (%)
76.6
Number of peaks
6162 (qval < 1E-05)

Base call quality data from DBCLS SRA