GSM1121660: Kyoto DGRC204406 hom input; Drosophila melanogaster; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Adult
Cell type
Ovary
NA
NA
Attributes by original data submitter
Sample
source_name
Drosophila melanogaster ovary
tissue
ovaries
strain
DGRC 204406 homozygous
antibody
none
sample type
ChIP input
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. ChIP was done as described in Ram et al. (2011) and Garber et al. (2012), with some modifications. Approximately fifty ovaries were dissected from heterozygous or homozygous flies into cold PBS and washed once with PBS. Ovaries were then fixed in 1.8% formaldehyde for 10 minutes, then quenched by adding glycine to 0.125M and immediately placed on ice. Tissue was then homogenized by douncing five times with pestle A (Kontes). Washed once with PBS supplemented with protease inhibitors (Roche) and pellet was flash frozen in liquid nitrogen. Pellets were then thawed on ice and resuspended in 1mL Lysis Buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1) and lysed for 10 minutes in ice. Chromatin was sheared to 200-800bp using a Branson sonifier (model S-450D). After clearing lysate by centrifugation, 9mLs of Dilution Buffer (0.01% SDS, 1.1%Triton X-100, 1.2mM EDTA, 16.7mM Tris-HCl, pH 8.1, 167mM NaCl) were added to the lysate and 5mLs of the lysate were incubated with a 50ul of an equal mixture of conjuagted protein A and G Dynabeads (Invitrogen). To conjugate beads, they first had been washed once in Blocking Buffer (1X PBS, 0.5% TWEEN 20, 0.5% BSA), then coupled for 1 hour at 4ºC with 5ug of H3K9me3 antibody (Abcam 8898) and finally washed twice with Blocking Buffer to remove excess antibody. Lysate and conjugated magnetic beads were rotated at 4ºC overnight. Beads were then resuspended in 200ul cold RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 14 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% DOC) and transfer to a 96-well plate. All further separation steps were performed in the 96-well plate magnet. Beads were washed five times with 200ul cold RIPA, two times with RIPA buffer supplemented with 500 mM NaCl, two times with LiCl buffer (10 mM TE, 250mM LiCl, 0.5% NP-40, 0.5% DOC), and once with TE (10Mm Tris-HCl pH 8.0, 1mM EDTA). Samples were eluted in 50 µl of 0.5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris HCl pH 8.0. The eluate was reverse cross-linked at 65ºC for 4 hours and then treated with 2ul of RNaseA (Roche, 11119915001) for 30 min followed by 2.5 µl of Proteinase K (NEB, P8102S) for two hours. Library preparation was done as indicated in Garber et al. (2012), but without automation. In brief, to purify DNA 120ul of Ampure XP beads (Agencourt) were added to the reverse cross-linked samples, mixed by pipetting and incubated for 2 minutes. Samples were then placed on the magnetic stand for 4 minutes to separate beads, followed by 2 washes with 70% ethanol and air dried for 4 minutes and eluted in 10mM Tris-HCl pH 8.0. Library was constructed by performing DNA end-repair, A-base addition, adaptor ligation and enrichment PCR. After each step DNA was purified by adding 20% PEG and 2.5 M NaCl to the reaction, to allow DNA to bind to Ampure XP beads already in the tube. Samples were not moved form their original well position, until after PCR enrichment. The libraries were sequenced on the Illumina HiSeq platform for 76 cycles in a pair-end run.