title: GSM1973501: Input; Xenopus laevis; ChIP-Seq
library_construction_protocol: Embryos (n= 35-90) were fixed in 1% formaldehyde for 30 minutes at the indicated developmental stage. Embryos were washed once in 125 mM glycine / 25% Marc's modified Ringer's medium (MMR) and twice in 25% MMR, homogenized on ice in sonication buffer (20 mM Tris•HCl, pH 8/10 mM KCl/1mM EDTA/10% glycerol/5 mM DTT/0.125% Nonidet P-40, and protease inhibitor cocktail (Roche)). Homogenized embryos were sonicated for 20 minutes using a Bioruptor sonicator (Diagenode). Sonicated extract was centrifuged at top speed in a cold table-top centrifuge and supernatants (ChIP extracts) were snap frozen in liquid nitrogen and stored at -20 degree C until used. Prior to assembling the ChIP reaction, the ChIP extract was diluted with IP buffer (50 mM Tris•HCl, pH 8/100 mM NaCl/2mM EDTA/1 mM DTT/1% Nonidet P-40, and protease inhibitor cocktail) and then incubated with 1-5 µg of antibody and 12.5 µl Prot A/G beads (Santa Cruz)  for an overnight  binding reaction on the rotating wheel in the cold room. The beads were sequentially washed, first with ChIP1 buffer (IP buffer plus 0.1% sodium deoxycholate),  then ChIP2 buffer (ChIP1 buffer with 500 mM NaCl final concentration), then ChIP3 buffer (ChIP1 buffer with 250 mM LiCl), then again with ChIP1 buffer, and lastly with TE buffer (10 mM Tris, pH 8/1 mM EDTA). The material was eluted in 1% SDS in 0.1M  sodium bicarbonate. Cross-linking was reversed by adding 16 µl  of 5M NaCl and incubating at 65°C for 4-5 hours. DNA was extracted using the Qiagen QIAquick PCR purification kit. ~ 10 ng input DNA was used  for sample preparation for high-throughput sequencing (according to manufacturer’s protocol). Libraries were prepared using the Kapa Hyper Prep kit (Kapa Biosystems) according to manufacturer's protocol and sequenced on an Illumina HiSeq 2000 or an Illumina NextSeq.
attributes.tag: GEO Accession
attributes.value: GSM1973501