GSM2238597: GlcNAc ChIP-seq on S2 cells using GalT purification; Drosophila melanogaster; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Unclassified
Antigen
Unclassified
Cell type
Cell type Class
Cell line
Cell type
S2
Source
Oregon R
Developmental Stage
late embryonic stage
Attributes by original data submitter
Sample
source_name
s2-cells-galt
biomaterial_provider
ATCC [D. Mel. (2), SL2] (ATCC® CRL-1963™)
tissue
Schneider's Drosophila Line 2 (SL2) cells
cell line
SL2
passages
10 to 12
chip antibody/selection
Purification with UDP-GalNAz and GalT labeling followed by click chemistry and streptavidin purification
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
~2x107 cells were fixed for 10 min at room temperature by gently mixing in 10 mL crosslinking solution (1% formaldehyde, 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), crosslinking was quenched by adding 2M glycine to a final concentration of 240 mM. The cells were sequentially collected by centrifugation at 700 x g for 10 min and washed trice with 10 mL ice-cold PBS. These cell pellets were sonicated in parallel to obtain chromatin fragments of ~200 to 700 bp, each in 4 mL sonication buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, Roche protease inhibitor cocktail) with a Sonic Dismembrator Model 500 (Fisher Scientific, 10 x 30 seconds on / 45 seconds off cycles, 50% power settings). After sonication, debris was removed by centrifugation at 4°C for 10 minutes at 13,000 rpm, and equal amount of 6M urea was added to the solution then incubated for 10 minutes at 4°C on a rotating wheel. The soluble chromatin was dialysed using a membrane with a molecular weight cut-off of 3.5 kDa, at 4°C against 2 L dialysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 4% glycerol) overnight. Insoluble debris was removed by quick centrifugation (7,500 rpm, 2 min). To label O-GlcNAcylated proteins with Ac4GalNAz, the Click-iT O-GlcNAc Enzymatic Labeling System was used (Invitrogen). Briefly, Gal-T1Y289L was incubated with proteins in labeling buffer (containing 20 mM HEPES, pH 7.9; 5 mM NaCl; 2% NP-40; 5.5 mM MnC2; 25 µM UDP-GalNAz), according to manufacturer's recommendations. Reaction was performed at 4°C under gentle agitation for 24 h. All reagents were provided in the kit. Once labeling achieved, proteins were chloroform/methanol precipitated. Note that the volume of each reagent was adjusted for higher proteins quantity, i.e., when 500 µg of proteins were labeled. The libraries were prepared according to the manufacturer's instructions using the NEBNext kit (E6200). Briefly, DNA was fragmented by sonication to a maximum of 300 bp. Next the ends of the fragments were repaired with a combination of fill-in reactions and exonuclease activity to produce blunt ends that were then tailed with an A-base. Illumina-specific Adaptors were ligated followed by removal of unligated adaptors using AMpure XP beads (Beckman Coulter). Finally a PCR with 12-15 cycles for both kits was performed to enrich final adaptor-ligated fragments. Quality and quantity were assessed on an Agilent Bioanalyzer Chip DNA 7500 or High Sensitivity. For all libraries, sequencing was performed on the MiSeq platform, using v2, 300-cycle reagent kits (Illumina).