organism: Mus musculus
description: A mouse cardiomyocyte cell line HL-1 was infected with an adenovector for expression of untagged GFP (Ad-GFP) and cultured for 24 hours. The cells were then irradiated with 254-nm UV (4,000 J/m2) and lysed, sonicated and digested with Turbo DNase (Ambion). FLAG-tagged protein was immunoprecipitated with Dynabeads Protein G (Invitrogen) and monoclonal anti-FLAG antibody M2 (SIGMA). Cross-linked and co-immunoprecipitated RNAs were then subjected to tRIP library preparation as described in EMBO Rep. 21: e49890 (2020). Briefly, the RNAs were digested with Shortcut RNase III (NEB) and ligated to a 5?f-preadenylated 3' linker-016, pAGATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGTCCATCTCGTATGCCGTCTTCTGCTTG-biotin, deadenylated with 5' Deadenylase (NEB), trimmed with Terminator Exonuclease (Lucigen) and purified with Quick-RNA Micro-Prep (Zymo) after Proteinase K digestion. The purified RNAs were reverse-transcribed with Superscript IV (Thermo) and a 1st-RT-primer CAAGCAGAAGACGGCAT, treated with Exonuclease I (NEB), RNase H (TaKaRa) and Terminal Transferase (NEB). The second strand was synthesized with MightyAmp DNA polymerase (TaKaRa) and a 2nd-str-primer CGACACGTCGCGTTTTTTTTTTVN. The library was then amplified with primers PCR-P5-full, AATGATACGGCGACCACCGAGATCTACACGCACGCTGTCCCGACACGTCGCGTTTT and PCR-P7-full-016, CAAGCAGAAGACGGCATACGAGATGGACGGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT and subjected to RNA-seq with a custom primer TB1005 GCACGCTGTCCCGACACGTCGCGTTTTTTTTTT and the standard Illumina platform.
identifiers.namespace: BioSample
identifiers.id: SAMD00406397
attributes.tag: sample_name
attributes.value: GFP
attributes.tag: tissue
attributes.value: HL-1 cells