title: GSM9246620: HT-29, IAV, FLAG-input, 3 [B831009]; Homo sapiens; RIP-Seq
library_construction_protocol: RIP was conducted using the EZ-Magna RIP (Cross-Linked) RNA-Binding Protein Immunoprecipitation Kit (Millipore) following the manufacturer's instructions. Briefly, cross-linked cell pellets were lysed in RIP lysis buffer, followed by incubation with RIP Buffer containing magnetic beads conjugated with anti-Z-NA antibody (clone Z22, Absolute Antibody) or anti-FLAG antibody (F1804, Sigma-Aldrich), at 4°C overnight. Samples were then incubated with proteinase K and immunoprecipitated RNAs were recovered by phenol:chloroform:isoamyl alcohol purification.  Ten percent of the cell lysate was used for total RNA extraction as the input, and the remaining ninety percent was used for immunoprecipitation. Libraries were prepared from RIP or input RNA using the TruSeq Stranded Total RNA Library Prep Kit according to the manufacturer's instructions (Illumina). Libraries were analyzed for insert size distribution using the 2100 BioAnalyzer High Sensitivity kit (Agilent), 4200 TapeStation D1000 ScreenTape assay (Agilent), or 5300 Fragment Analyzer NGS fragment kit (Agilent). Libraries were quantified using the Quant-iT PicoGreen ds DNA assay (Thermo Fisher Scientific) or by low pass sequencing with a MiSeq nano kit (Illumina). Paired-end 150 cycle sequencing was performed on a NovaSeq 6000 (Illumina).