title: GSM1180940: XBRA20.1; Xenopus tropicalis; ChIP-Seq
library_construction_protocol: Cross-linked chromatin was extracted with E1, E2 and E3 (Lee et al., 2006 , PMID:17406303). Chromatin was solubilised and sheared to fragments of 100-500bp peaking around 200 bp. ChIP was performed with ChIP-grade antibodies coupled to Dynabeads M-280. Beads were extensively washed with 10 times pre-chilled RIPA buffer (50 mM Hepes-KOH pH7.5, 500 mM LiCl, 1mM EDTA, 1% Igepal CA-630, 0.7% (w/v) Na-deoxycholate) and twice in pre-chilled TEN (10 mM Tris pH8.0, 1 mM EDTA, 150 NaCl). Bound chromatin was eluted from the beads with elution buffer (50 mM Tris pH8.0, 1 mM EDTA pH8.0, 1% SDS) at 65C for 30 min. ChIP and input samples were supplemented with 1/20 volume of 5 M NaCl before incubating them for 6-15 h at 65C to reverse cross-links. One volume of TE pH 8.0 and 200 µg/ml of CONCERT RNase A (Invitrogen) was added to the samples for a 1-h incubation at 37C. The samples were further treated with 200 µg/ml of proteinase K (Ambion) for 2-4 h at 55C. Eventually, the DNA fragments were purified with phenol:chloroform:isoamylalcohol and precipitated in ethanol for sequencing. The air-dried DNA pellet was dissolved in 32 µl of molecular-grade water. The amount of DNA was quantified using a Qubit fluorometer (Invitrogen) following the manufacturer's instructions. The yield of co-immunoprecipitated DNA was about 10 ng, which created robust libraries. The single-end library was generated based on the manufacturer's instructions (Illumina) with minor modifications. DNA fragments were end-repaired, ligated to adaptors at a final dilution of 1:480, size-selected to 200-300 bp by E-gel (Invitrogen) and PCR (18 cycles) amplified. The DNA Clean & Concentrator-5 kit (Zymo Research) was used to clean up reactions. DNA was eluted from the column at 50C. Upon solid phase reversible immobilisation (SPRI) purification, the integrity of the library was checked on the Agilent 2100 Bioanalyzer using an Agilent DNA 1000 chip. ChIP-Seq libraries were sequenced for 40 cycles on the Illumina Genome Analyser IIx.