title: GSM7783160: WT_rip; Mus musculus; RIP-Seq
library_construction_protocol: Four brains of P4 mice brain were lysed in 8 ml Lysis Buffer (10 mM HEPES pH 7.6, 2 mM EDTA, 150 mM NaCl, 0.5% NP-40, 0.5 mM DTT, cocktail, 400 U/ml RNase inhibitor). Tissue was fully cleaved with a homogenizer and lysates were centrifuged at 12,500rpm for 60 min at 4 °C. Then the lysate (4.0 mg/ml) were incubated with HA-tag Magnetic Beads (#88836, Thermo Scientific, USA) at 4 °C overnight. After washing, the beads collected and resuspended in 1× MN reaction buffer with micrococcal nuclease at 37 °C for 10 min were washed and incubated with 1×PK buffer containing Proteinase K. RNA were extracted by phenol-chloroform method and then used for RNA library construction (Illumina). RIP products were reversed transcription into cDNA sequencing library using Invitrogen SuperScript™ II Reverse Transcriptase kit (CA, USA, 1896649). RNA-seq libraries were generated with the NEBNext Ultra™ RNA library Prep Kit (NEB E7770S). The libraries were subjected to quality validation using the Agilent Bioanalyzer 2100, and sequenced using Illumina platforms via a 2x150bp paired-end sequencing protocol