GSM1540987: d0 CLIP rep2 [mUV]; Mus musculus; RIP-Seq
CLIP-seq: Nuclei were isolated and lysed. Nuclear lysates were DNase-treated, then fragmented by sonication to an RNA size of 150-200 nt. Cleared lysates were incubated overnight with 15 uL of Anti-FLAG M2 Magnetic Beads. After stringent washing and DNase treatment, CLIP-tags on beads were radiolabelled with [γ-32P]ATP, run on Bis-Tris SDS-PAGE in MOPS buffer, and transferred to nitrocellulose membrane. Membrane fragments containing CLIP signal and corresponding positions on control lanes were excised, and RNA was eluted by proteinase K, followed by Trizol extraction and ethanol precipitation. RNA-seq: Nuclear lysate was removed from CLIP protocol before sonication and treated with proteinase K and reprecipitated in the same way as CLIP RNA eluted from membrane. RNA was depleted of ribosomal RNA with RiboMinus Eukaryote System (Ambion A15026), treated with TURBO DNase, and cleaned up with RNeasy MinElute columns (Qiagen 74204). RNA was fragmented in Superscript III first strand synthesis buffer before first strand cDNA synthesis. ChIP-seq: Cells were crosslinked with 1% formaldeyde for 10 min, then nuclei were isolated and lysed. Chromatin was sheared by sonication to a size of ~1-2kb. Cleared lysates were incubated overnight with CTCF antibodies, with 10% saved as input, then captured with Dynabeads Protein G. After stringent washes, ChIP DNA was eluted from beads, and together with input chromtain was reverse-crosslinked with proteinase K. DNA was purified by phenol/chloroform extraction and ethanol precipitation. CLIP-seq: Library was constructed from CLIP RNA using the NEBNext Small RNA Library Prep set (New England Biolabs E7330), size-selected and cleaned up of primer/adaptor-dimers using Agencourt AMPure XP beads. RNA-seq: Fragmented RNA was used for first strand cDNA synthesis with the addition of actinomycin D. Second strand cDNA synthesis was performed with NEBNext mRNA Second Strand Synthesis Module (E6111) in the presence of dUTP to preserve directionality. Double-stranded cDNA was then used to generate RNA-seq libraries using the NEBNext ChIP-Seq Library Prep Master Mix Set (E6240). ChIP-seq: Library was constructed from ChIP DNA using the NEBNext ChIP-Seq Library Prep Master Mix Set.