Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
tin

Cell type

Cell type Class
Embryo
Cell type
13-14h embryos
NA
NA

Attributes by original data submitter

Sample

Alias
E-MTAB-4584:Tin-GFP
Broker name
ArrayExpress
Description
Protocols: Embryos were collected from 4 large cylindrical population cages, each seeded with ~70 g of newly eclosed flies (representing ~70000 flies). After performing three 1 h pre-lays, 3 h embryo collections using 3 standard molasses plates (3g agar, 3g sugar, 30mL apple juice, 100mL water, 1mL propionic acid) streaked with yeast paste) by cages typically yielded 3-5 g dry weight of tightly staged embryos. Cages were maintained at 25C and 60% of hygrometry. Embryos were washed and transfer into large sieves. After dechorionation (2.5 min in 400 ml 3% NaOCl (50% bleach)) embryos were transferred to a Nitex membrane (5x5 cm) to be dried with a paper towel. Then they were placed into a 50 ml Falcon tube with 9.5ml Cross-linking solution (50 mM Hepes, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, pH 8), 485 microl 37% Formaldehyde and 30ml n-Heptane and shaked vigorously at room temperature (20-25C) for 15 min. They were washed once with 30 ml PBS/Glycin/Triton (125 mM glycine, 0.1% Triton in PBS), once with 50 ml ice-cold PBT and suspended in 10 ml PBT (PBS, 0.1% Triton). After drying, cross-linked embryos were freezed in liquid nitrogen and stored at -80C. Nuclei were extracted as described by Bonn and colleagues (Cell type specific chromatin immuno precipitation from multicellular complex samples using BiTS-ChIP, Nat Prot, 2012). Then, nuclei were dissociated by passing them ten times through a 20-G needle and then ten times through a 22-G needle using a 5-ml syringe and passed through a square of 20um Sefar Nitex membrane. First antibody used to stain nuclei was a mouse anti-GFP (Abfinity recombinant rabbit monoclonal G10362) which is expressed specifically in cardioblasts, at 1:100 for 1h nutating at 4C. To remove most of the free primary antibody we added 6 ml of PBTB, spin for 2 min at 1,000g and discard the supernatant. The fluorescent antibody was an anti-mouse Alexa Fluor 488 (Alexa488) at 1:100 in 3ml of PBTB and incubated for 1h while nutating at 4C. MoFlo cell sorter (Beckman Coulter Inc.) equipped with a 70microm nozzle was used during flow cytometric analysis and cell sorting of GFP positive embryonic nuclei. More details can be found in the manuscript methods. After sorting, the nuclei were centrifuged at 3500 g for 10 min and the pellet suspended in 300 microl RIPA buffer and transferred into a 1.5 ml tube (RIPA: 140mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, Roche proteinase inhibitors).After incubation for 10 min on ice, the chromatin was sheared into 200 bp fragments using a Diagenode BioRuptor (18 cycles, high intensity, 30s on/off intervals, ice-cold water cooled continually at 4C).The sample was centrifuged for 2 min at 18000 g and the supernatant was transferred to a low-binding tube. While the majority of sheared chromatin was subsequently snap-frozen in liquid nitrogen, a small aliquot was used to measure DNA concentration and fragment size after reverse cross-linking (by addition of 1microL 20mg/ml proteinase K, incubate at 37C 2h and overnight at 65C). 3gr of embryos typically yielded 0.5microg of stage and tissue specific chromatin. Immunoprecipitations (IP) conditions were therefore downscaled in order to allow multiple IP. Compared to classical ChIP protocol (Bonn), the main modifications were a downscaling of beads and antibodies quantities and an adjustment of reaction volumes. For each reaction, 500ng of chromatin were bring to 500microL TE and 500microL RIPA buffer (140 mM NaCl, 1 mM EDTA, 1% (vol/vol) Triton X-100, 0.1% (wt/vol) SDS, 0.1% (wt/vol) sodium deoxycholate and 10 mM Tris-HCl (pH 8.0)) supplemented with protease inhibitor. Input sample (10microL) was put aside and stored at 4C. 1microL of commercially available antibodies against H3K4me3 (ab71998) and H3K27ac (ab4729) (abcam) were added to chromatin. At the same time, for each precipitation 10microL of 50% ProteinA Sepharose (PAS) suspension (Protein A Sepharose CL4B, Sigma) was washed once with 1 mL RIPA buffer + 1mg/mL BSA, spinned 1000g 2' and SN discarded. Beads were suspended in 1mL of RIPA buffer + 1mg/mL BSA O/N at 4C by mixing gently. Immune-complexes were purified by adding pre-absorbed beads for 3h nutating at 4C then washed (10 min each) once with RIPA, 4 times with RIPA 500 (500mM NaCl, 1 mM EDTA, 1% (vol/vol) Triton X-100, 0.1% (wt/vol) SDS, 0.1% (wt/vol) sodium deoxycholate and 10 mM Tris-HCl (pH 8.0)), once with LiCL (250 mM LiCl, 1 mM EDTA, 0.5% (vol/vol) IGEPAL CA-630, 0.5% (wt/vol) sodium deoxycholate and 10 mM Tris-HCl (pH 8.0)) and twice with TE buffer. IPed DNA was suspended in 100microL of TE and treated with RNAse (50microg/ml) 30 min at 37C. Then, samples were adjusted to 0.5%SDS, 0.5 mg/mL proteinase K and incubated 2h at 37C and overnight at 65C. DNA samples were purified with Phenol:Chloroform:Isoamylalcohol (25:24:1) and 2ml PhaseLock gel tube (Heavy). Purified DNA was supplemented 10microL glycogen 5mg/mL as carrier, 20microL NaOAc 3M pH 5.3 and 550microL of 100% EtOH and placed at -80C at least 30 minutes to precipitate. After spinning 30 min at 14,000g at 4C, wash with 70% EtOH. After spinning 10 min, SN was removed and pellet air-dried at RT for at least 10 min. DNA was suspended in 30microL TE. The Extraction is part of IP described in the sample's ChIP protocol We used NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina with minor midifications. Notably, we increased the time of ligation from 15 minutes to 45 minutes. It is generally advisable to use the fewest PCR cycles possible, and we routinely use 15-18 cycles. The resulting library eluate should be in excess of 2 ng/microl, and we routinely have concentrations of 20-50 ng/microl. We find that we obtain much better results by performing Ampure magnetic beads isolation after PCR amplification.
ENA checklist
ERC000011
INSDC center name
EMBL
INSDC first public
2017-01-10T17:05:11Z
INSDC last update
2016-04-18T14:12:01Z
INSDC status
public
SRA accession
ERS1119250
Sample Name
ERS1119250
Title
Tin-GFP
age
10-13h
developmental stage
stage13-14
initial time point
egg laying
organism part
embryonic cardiac cells
sex
mixed sex
strain
TinC-GFP

Sequenced DNA Library

library_name
BITSHeart_Heart5_K27ac_10-13h
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Embryos were collected from 4 large cylindrical population cages, each seeded with ~70 g of newly eclosed flies (representing ~70000 flies). After performing three 1 h pre-lays, 3 h embryo collections using 3 standard molasses plates (3g agar, 3g sugar, 30mL apple juice, 100mL water, 1mL propionic acid) streaked with yeast paste) by cages typically yielded 3-5 g dry weight of tightly staged embryos. Cages were maintained at 25C and 60% of hygrometry. Embryos were washed and transfer into large sieves. After dechorionation (2.5 min in 400 ml 3% NaOCl (50% bleach)) embryos were transferred to a Nitex membrane (5x5 cm) to be dried with a paper towel. Then they were placed into a 50 ml Falcon tube with 9.5ml Cross-linking solution (50 mM Hepes, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, pH 8), 485 microl 37% Formaldehyde and 30ml n-Heptane and shaked vigorously at room temperature (20-25C) for 15 min. They were washed once with 30 ml PBS/Glycin/Triton (125 mM glycine, 0.1% Triton in PBS), once with 50 ml ice-cold PBT and suspended in 10 ml PBT (PBS, 0.1% Triton). After drying, cross-linked embryos were freezed in liquid nitrogen and stored at -80C. Nuclei were extracted as described by Bonn and colleagues (Cell type specific chromatin immuno precipitation from multicellular complex samples using BiTS-ChIP, Nat Prot, 2012). Then, nuclei were dissociated by passing them ten times through a 20-G needle and then ten times through a 22-G needle using a 5-ml syringe and passed through a square of 20um Sefar Nitex membrane. First antibody used to stain nuclei was a mouse anti-GFP (Abfinity recombinant rabbit monoclonal G10362) which is expressed specifically in cardioblasts, at 1:100 for 1h nutating at 4C. To remove most of the free primary antibody we added 6 ml of PBTB, spin for 2 min at 1,000g and discard the supernatant. The fluorescent antibody was an anti-mouse Alexa Fluor 488 (Alexa488) at 1:100 in 3ml of PBTB and incubated for 1h while nutating at 4C. MoFlo cell sorter (Beckman Coulter Inc.) equipped with a 70microm nozzle was used during flow cytometric analysis and cell sorting of GFP positive embryonic nuclei. More details can be found in the manuscript methods. After sorting, the nuclei were centrifuged at 3500 g for 10 min and the pellet suspended in 300 microl RIPA buffer and transferred into a 1.5 ml tube (RIPA: 140mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, Roche proteinase inhibitors).After incubation for 10 min on ice, the chromatin was sheared into 200 bp fragments using a Diagenode BioRuptor (18 cycles, high intensity, 30s on/off intervals, ice-cold water cooled continually at 4C).The sample was centrifuged for 2 min at 18000 g and the supernatant was transferred to a low-binding tube. While the majority of sheared chromatin was subsequently snap-frozen in liquid nitrogen, a small aliquot was used to measure DNA concentration and fragment size after reverse cross-linking (by addition of 1microL 20mg/ml proteinase K, incubate at 37C 2h and overnight at 65C). 3gr of embryos typically yielded 0.5microg of stage and tissue specific chromatin. Immunoprecipitations (IP) conditions were therefore downscaled in order to allow multiple IP. Compared to classical ChIP protocol (Bonn), the main modifications were a downscaling of beads and antibodies quantities and an adjustment of reaction volumes. For each reaction, 500ng of chromatin were bring to 500microL TE and 500microL RIPA buffer (140 mM NaCl, 1 mM EDTA, 1% (vol/vol) Triton X-100, 0.1% (wt/vol) SDS, 0.1% (wt/vol) sodium deoxycholate and 10 mM Tris-HCl (pH 8.0)) supplemented with protease inhibitor. Input sample (10microL) was put aside and stored at 4C. 1microL of commercially available antibodies against H3K4me3 (ab71998) and H3K27ac (ab4729) (abcam) were added to chromatin. At the same time, for each precipitation 10microL of 50% ProteinA Sepharose (PAS) suspension (Protein A Sepharose CL4B, Sigma) was washed once with 1 mL RIPA buffer + 1mg/mL BSA, spinned 1000g 2' and SN discarded. Beads were suspended in 1mL of RIPA buffer + 1mg/mL BSA O/N at 4C by mixing gently. Immune-complexes were purified by adding pre-absorbed beads for 3h nutating at 4C then washed (10 min each) once with RIPA, 4 times with RIPA 500 (500mM NaCl, 1 mM EDTA, 1% (vol/vol) Triton X-100, 0.1% (wt/vol) SDS, 0.1% (wt/vol) sodium deoxycholate and 10 mM Tris-HCl (pH 8.0)), once with LiCL (250 mM LiCl, 1 mM EDTA, 0.5% (vol/vol) IGEPAL CA-630, 0.5% (wt/vol) sodium deoxycholate and 10 mM Tris-HCl (pH 8.0)) and twice with TE buffer. IPed DNA was suspended in 100microL of TE and treated with RNAse (50microg/ml) 30 min at 37C. Then, samples were adjusted to 0.5%SDS, 0.5 mg/mL proteinase K and incubated 2h at 37C and overnight at 65C. DNA samples were purified with Phenol:Chloroform:Isoamylalcohol (25:24:1) and 2ml PhaseLock gel tube (Heavy). Purified DNA was supplemented 10microL glycogen 5mg/mL as carrier, 20microL NaOAc 3M pH 5.3 and 550microL of 100% EtOH and placed at -80C at least 30 minutes to precipitate. After spinning 30 min at 14,000g at 4C, wash with 70% EtOH. After spinning 10 min, SN was removed and pellet air-dried at RT for at least 10 min. DNA was suspended in 30microL TE. The Extraction is part of IP described in the sample's ChIP protocol We used NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina with minor midifications. Notably, we increased the time of ligation from 15 minutes to 45 minutes. It is generally advisable to use the fewest PCR cycles possible, and we routinely use 15-18 cycles. The resulting library eluate should be in excess of 2 ng/microl, and we routinely have concentrations of 20-50 ng/microl. We find that we obtain much better results by performing Ampure magnetic beads isolation after PCR amplification.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

dm3

Number of total reads
4686358
Reads aligned (%)
37.6
Duplicates removed (%)
41.1
Number of peaks
1779 (qval < 1E-05)

dm6

Number of total reads
4686358
Reads aligned (%)
31.6
Duplicates removed (%)
48.1
Number of peaks
1424 (qval < 1E-05)

Base call quality data from DBCLS SRA