title: GSM9053720: ∆wblA biological replicate #15 + WblA_Flag 16 hour; Streptomyces venezuelae; ChIP-Seq
library_construction_protocol: Lysates were cleared by centrifugation at 17,005×g for 20 minutes at 7 °C. Anti-FLAG M2 beads (100 µL) were pre-washed with 1 mL IPP150 buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% NP-40) and incubated briefly with rotation at 4 °C before pelleting at 226×g for 5 minutes. The cleared lysate was adjusted by adding per mL: 10 µL of 1 M Tris-HCl pH 8.0, 20 µL of 5 M NaCl, and 10 µL of 10% NP-40. After brief centrifugation (226×g, 5 min), the lysate was added to the prepared beads and rotated overnight at 4 °C. A 20 µL aliquot was retained for DNA fragmentation assessment. Beads were washed five times with 1 mL IPP150 buffer (inverting 1–2 minutes, then centrifugation at 226×g for 5 minutes at 7 °C), followed by two washes with 1× TE buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA). All supernatant was carefully removed after the final wash. Elution was performed by incubating beads with 150 µL of elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) at 65 °C for 15 minutes. After centrifugation (226  RCF, 5 min), a second elution was performed with 100 µL of 1× TE + 1% SDS at 65 °C for 5 minutes. Eluates were pooled. Cross-links were reversed by incubating pooled eluates at 65 °C overnight. DNA was purified using a QIAquick PCR purification kit with 2.5 volumes of PB buffer, followed by two washes with PE buffer. Columns were spun dry and DNA was eluted twice in 20 µL dH₂O. DNA concentrations were measured with Qubit and samples were stored at –80 °C. DNA was quatified using a Qubit flourometer with a High Sensitivity dsDNA kit and shipped on dry ice to Novogene. Once samples arrived at Novogene they were treating using their standard protocols to repaired the DNA fragments , A-tailed and further ligated with Illumina adapter. The final DNA library was obtained by size selection and PCR amplification.The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount required