title: GSM5417957: 1-222mut_PAPI-CLIP=seq_rep1; Bombyx mori; RIP-Seq
library_construction_protocol: 10 cm dish BmN4 cells transfected Papi-Flag or Papi1-222-Flag were used for each iCLIP experiment. For UV crosslinking, BmN4 cells were washed once with ice-cold PBS. The UV crosslinking was performed with irradiance of 200 mJ/cm2 at 254 nm. Cells were pelleted and lysed with lysis buffer [20 mM HEPES (pH7.3), 150 mM sodium chloride, 1 mM EDTA, 1 mM DTT, 0.5% Triton X-100, 2 μg/mL pepstatin, 2 μg/mL leupeptin, 0.5% aprotinin]. RNA–protein complexes were immunoprecipitated using 12 μg of anti-FLAG M2 antibody and 120 μL of Dynabeads Protein G at 4 °C for 2 h. Beads were washed three times with wash buffer [20 mM HEPES (pH7.3), 300 mM sodium chloride, 1 mM DTT, 0.5% Triton X-100, 2 μg/mL pepstatin, 2 μg/mL leupeptin, 0.5% aprotinin], followed by partial RNA digestion with 1 U/μL RNase T1 for 15 min at room temperature. Beads were washed three times with high-salt wash buffer [20 mM HEPES (pH7.3), 500 mM sodium chloride, 1 mM DTT, 0.5% Triton X-100, 2 μg/mL pepstatin, 2 μg/mL leupeptin, 0.5% aprotinin]. After 3' end RNA dephosphorylation with T4 PNK, the RNAs were ligated at their 3' end s to a preadenylated RNA adaptor with T4 RNA ligase2, truncated KQ (New England Biolabs), and then radioactively labeled. The samples were analyzed by NuPAGE gel electrophoresis (Life Technologies) and RNA–protein complexes were transferred to nitrocellulose. After cutting out the region of nitrocellulose containing RNA–protein complexes, RNAs were removed from the membrane by Proteinase K (Roche) digestion. Extracted samples were processed for reverse transcription, cDNA purification, and cDNA circularization as previously described (Flynn et al., 2015). Libraries were then amplified with Q5 DNA polymerase (New England Biolabs) and size-selected via polyacrylamide gel electrophoresis.