GSM2830903: NF-H_P_2; Homo sapiens; RIP-Seq
Differentiated neural stem cells were detached by trypsin (0.5%, Life Technologies) and collected in RPMI medium with 10% FCS (Life Technologies). Cells were pelleted by centrifugation and washed twice in ice-cold PBS (14190-094) before being lysed in four cell-pellet volumes of either Lysis Buffer 1 [20 mM Tris-HCl pH 7.5, 150 mM NaCl, 3 mM EDTA, 1% Triton X-100, 0.5% Na-Deoxychoalte, 1X protease inhibitors cocktail (Roche), 1 mM DTT Lysed cells were sonicated (Bioruptor, Diagenode) at maximum setting for 5 seconds on ice and centrifuged at 21.000 x g for 30 min at 4oC. The supernatant was filtered through a 0.1 µm syringe filter (Santa Cruz, sc-358809) into new tubes and the protein concentration determined with the BCA kit (Thermo Fisher) according to the manufacturer's instructions. Lysates were diluted in Lysis Buffer-1 to 2-4 µg/µl and used for IP. Immunoprecipitated RNA from human neuronal lysate were purified on silica columns, and include a DNase I treatment step to remove DNA contaminants. RNA were eluted from the columns and EtOH precipitated before being used for library preparation. Library preparation were prepared from RNA using the NEBNext Ultra II Directional RNA Library Prep Kit and Indexing primers (E7760, and E7335, respectively, both NEB) according to the manufacturer's instructions. Libraries were pooled and pair-end sequenced (2x75 bp) on the NextSeq 500 platform (Illumina).
GEO Accession
GSM2830903