GSM8459897: ctrl mice, rep1, HA IP; Mus musculus; RIP-Seq
Retinas from the same animal were combined and homogenized in lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM KCl, 12 mM MgCl2, 1% NP-40, 0.4 unit/ml RNase inhibitor, 1 mM DTT, 100 μg/ml cycloheximide) with a dounce homogenizer. Lysates were incubated with 1 µg/µl heparin for 2 minutes on ice prior to centrifugation. The lysates were spun down at 10,000g for 10 minutes at 4C and the supernatant was used for immunoprecipitation using an antibody against HA-tag with Dynabeads protein G for overnight at 4C. 1.5% of lysate was saved as an input. After extensive washes with cold washing buffer (50 mM Tris-HCl pH 7.4, 300 mM KCl, 12 mM MgCl2, 1% NP-40, 0.5 mM DTT, 100 μg/ml cycloheximide), immunoprecipitated RNA and input RNA were purified with a RNeasy Micro Kit (Qiagen) following the manufacturer's instructions and eluted in 10 µl of water. 2 µl of purified RNA were used for sequencing library preparation using the Smart-seq3 protocol with modifications. After reverse transcription followed by PCR amplification, 25 ng of cDNA was used for the Tn5 tagmentation reaction. Tagmentated cDNA was amplified with 8 cycles of PCR using primers containing indexes and Illumina adaptors.