Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
Lint-1

Cell type

Cell type Class
Cell line
Cell type
Kc167
Source
e/se
Developmental Stage
dorsal closure stage

Attributes by original data submitter

Sample

Alias
E-MTAB-855:KC_dLint1
Broker name
ArrayExpress
CellLine
KC
Description
Protocols: D. melanogaster cell lines were maintained under standard conditions. ChIPs were performed as described in the Upstate Biotechnology ChIP assay protocol. 100*10^6 Kc cells were fixed in 1% formaldehyde for 10 min at RT. Fixation was stopped by the addition of 240 mM glycine. Cells were harvested, washed in ice cold PBS, resuspended in 1 ml SDS-Lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS, protease inhibitors) and incubated for 10 min on ice. Lysates were sonicated using a Bioruptor (Diagenode) to obtain an average fragment length of 0.5 kb and centrifuged (at 4 degrees C, 15 min, 13000 rpm). Shearing of the DNA was analyzed by agarose gel electrophoresis following reversal of crosslinks. The supernatant (chromatin) was subjected to ChIP analysis. 8 microliter of anti-dLint-1 #1 were used per ChIP. 140 microliter of chromatin were used per ChIP, diluted 10x with IP-buffer (16.7 mM Tris-HCl, pH 8.0, 16.7 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, protease inhibitors) and pre-cleared with 40 microliter of pre-blocked ProtG beads (1mg/ml BSA, 4 h) (GE Healthcare) for 30 min with rotation. Incubation with antibodies was performed overnight, prior to incubation for 2 h with 35 microliter of 1:1 ProtG slurry at 4 degrees C. Precipitates were serially washed for 10 min, three times with low salt wash buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton-X-100), three times with high salt wash buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton-X-100), once with LiCl wash buffer (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate), once with TE buffer. Immunoprecipitates were eluted twice with 250 microliter elution buffer (1% SDS, 0.1 M NaHCO3) for each 15 min at RT and crosslinks were reversed by addition of 20 microliter 5 M NaCl and heating at 65 degrees C overnight. Following addition of 10 microliter of 0.5 M EDTA, 20 microliter 1 M Tris-HCl, pH 6.5 and 2 microliter of 10 mg/ml Proteinase K samples were incubated for 1 h at 45 degrees C. DNA was purified with peqGOLD Cycle-Pure Kit (Peqlab) Generation of Lint-1 specific antibodies The C-terminus (aa 302-602) of dLint-1 was fused to GST by cloning into pGex4T1 vector. The recombinant protein was expressed in E. coli BL21 according to standard procedures. The GST-Lint-1-C-term fusion protein was purified via affinity chromatography using a GSTrap FF column (GE Healthcare) and subsequent ion exchange chromatography using a HiTrap SP HP column (GE Healthcare) on an Akta purifier system (GE Healthcare) according to the manufacturer's instructions. For immunization two rabbits (serum #1 and #2) were injected with 0.5 mg of purified GST-Lint-1-C-term fusion protein each (Peptide Speciality Laboratories, Heidelberg, Germany). The specificity of antibodies was verified by RNA interference in Kc cells and subsequent Western blot analysis of nuclear extracts.
INSDC center alias
Helmholtz Centre for Infection Research
INSDC center name
Helmholtz Centre for Infection Research
INSDC first public
2012-06-27T13:31:32Z
INSDC last update
2018-03-08T15:32:18Z
INSDC status
public
SRA accession
ERS071647
Sample Name
ERS071647
Title
KC_dLint1

Sequenced DNA Library

library_name
CHIP_KC_dLint1
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
D. melanogaster cell lines were maintained under standard conditions. ChIPs were performed as described in the Upstate Biotechnology ChIP assay
protocol. 100*10^6 Kc cells were fixed in 1% formaldehyde for 10 min at RT.
Fixation was stopped by the addition of 240 mM glycine. Cells were harvested,
washed in ice cold PBS, resuspended in 1 ml SDS-Lysis buffer (50 mM Tris-HCl,
pH 8.0, 10 mM EDTA, 1% SDS, protease inhibitors) and incubated for 10 min on
ice. Lysates were sonicated using a Bioruptor (Diagenode) to obtain an average
fragment length of 0.5 kb and centrifuged (at 4 degrees C, 15 min, 13000 rpm).
Shearing of the DNA was analyzed by agarose gel electrophoresis following
reversal of crosslinks. The supernatant (chromatin) was subjected to ChIP
analysis. 8 microliter of anti-dLint-1 #1 were used per ChIP. 140 microliter of
chromatin were used per ChIP, diluted 10x with IP-buffer (16.7 mM Tris-HCl, pH
8.0, 16.7 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, protease
inhibitors) and pre-cleared with 40 microliter of pre-blocked ProtG beads
(1mg/ml BSA, 4 h) (GE Healthcare) for 30 min with rotation. Incubation with
antibodies was performed overnight, prior to incubation for 2 h with 35
microliter of 1:1 ProtG slurry at 4 degrees C. Precipitates were serially
washed for 10 min, three times with low salt wash buffer (20 mM Tris-HCl pH
8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton-X-100), three times with high
salt wash buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1%
Triton-X-100), once with LiCl wash buffer (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM
EDTA, 1% NP-40, 1% sodium deoxycholate), once with TE buffer.
Immunoprecipitates were eluted twice with 250 microliter elution buffer (1%
SDS, 0.1 M NaHCO3) for each 15 min at RT and crosslinks were reversed by
addition of 20 microliter 5 M NaCl and heating at 65 degrees C overnight.
Following addition of 10 microliter of 0.5 M EDTA, 20 microliter 1 M Tris-HCl,
pH 6.5 and 2 microliter of 10 mg/ml Proteinase K samples were incubated for 1 h
at 45 degrees C. DNA was purified with peqGOLD Cycle-Pure Kit (Peqlab)

Generation of Lint-1 specific antibodies
The C-terminus (aa 302-602) of dLint-1
was fused to GST by cloning into pGex4T1 vector. The recombinant protein was
expressed in E. coli BL21 according to standard procedures. The
GST-Lint-1-C-term fusion protein was purified via affinity chromatography using
a GSTrap FF column (GE Healthcare) and subsequent ion exchange chromatography
using a HiTrap SP HP column (GE Healthcare) on an Akta purifier system (GE
Healthcare) according to the manufacturer's instructions. For immunization two
rabbits (serum #1 and #2) were injected with 0.5 mg of purified
GST-Lint-1-C-term fusion protein each (Peptide Speciality Laboratories,
Heidelberg, Germany). The specificity of antibodies was verified by RNA
interference in Kc cells and subsequent Western blot analysis of nuclear
extracts.

Sequencing Platform

instrument_model
Illumina Genome Analyzer IIx

dm3

Number of total reads
20615763
Reads aligned (%)
73.7
Duplicates removed (%)
53.0
Number of peaks
8743 (qval < 1E-05)

dm6

Number of total reads
20615763
Reads aligned (%)
73.3
Duplicates removed (%)
58.1
Number of peaks
5965 (qval < 1E-05)

Base call quality data from DBCLS SRA