GSM3426145: old yw ChIP rep1; Drosophila melanogaster; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Unclassified
Antigen
Unclassified
Cell type
Cell type Class
Adult
Cell type
Adult body
NA
NA
Attributes by original data submitter
Sample
source_name
Whole Body
tissue
Whole Body
developmental
35 days after eclosion
condition
old
Sex
female
genotype
yw;+;+
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
~200 female flies were anesthetized with Flynap (Carolina, Burlington, NC) and ground into a powder in liquid nitrogen. Crosslinking was allowed for 20 minutes in 1X PBS with 1% paraformaldehyde before quenched with glycine. Chromatin was washed several times with 1X PBS supplemented with protease inhibitor. Pellets were washed once with cold cell lysis buffer (5mM HEPES pH7.6, 100mM NaCl, 1M EDTA,0.5% NP-40, ddH2O, 0.1% PIC). Buffer was removed and samples were snap frozen in liquid nitrogen and stored at -80°C to synchronize experiments. Samples were thawed and treated with nuclear lysis buffer (50mM HEPES pH7.6, 10mM EDTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, ddH2O, 0.1% PIC) and incubated at 4°C for 10 minutes. Chromatin was sheared for 500bp using Branson digital sonifier 250, using 30%, with 30 seconds on 30 seconds 0ff for 5 cycles. Supernatant was snap frozen and stored at -80°C. Immunoprecipitation was carried out using Protein-G SureBeads (BioRad Hurcules, CA, USA). Beads were washed once with 1X PBS prior to blocking with 1X PBS and 0.5% BSA for 20 minutes at 4°C. Samples were precipitated with affinity purified anti-dFOXO antibody (Tatar Lab). Samples were reverse crosslinked at 65°C for 12 hours. DNA size selection and library prep were done using NEBNext Ultra II DNA library prep kit for Illumina an indexed using NEBNext multiplex oligos for Illumina (Primer set 1) (New England BioLabs, Ipwsich MA). DNA from either ChIP or input samples was mixed with AMPure XP beads (**) to select for a final library size of 320bp. Samples were diluted to a final concentration of 2nM for Illumina sequencing on Illumina 3000 (Illumina, San Diego CA) with single-end 50 bp reads format