GSM8031371: ac4c_GV_2; Mus musculus; OTHER
LACE-seq experiments were carried out according to the published protocol. In short, 50 oocytes were collected in 1.5 mL Eppendorf LoBind microcentrifuge tubes and quickly spun down to the bottom with a mini centrifuge. Samples were lysed on ice using 50 μl wash buffer for 10 min. Next, 1 μl SUPERase·In RNase inhibitor (Ambion, AM2696) and 4 μl RQ1 DNase (Promega, M6101) were applied to the lysate and incubated at 37 °C for 3 min. After snap-chilling the tube on ice for 3 min, 2 μg ac4c antibody and rotated at 4 °C for 1 h. Next, the samples were irradiated twice with UV-C light on the ice at 400 mJ, then 10 μl protein A/G beads were added to the sample and rotated for 2 h at 4 °C. After extensive wash steps, the immunoprecipitated RNAs were fragmented, and experienced 3′-dephosphorylation and linker ligation, followed by reverse transcription on-beads. Then, first-strand cDNAs were derived from protein A/G beads and further captured by streptavidin C1 beads for pre-PCR and added 3'-cDNA linker to produce dsDNA as the template for in vitro transcription (IVT). IVT products were purified by the removals of the DNA template using Turbo DNase and further purified by Agencourt RNA Clean beads based on the instructions of the manufacturer. Then, linear amplified RNA was transformed into cDNA and amplified by PCR with P7 and barcoded P5 index primers. Final PCR products with a size of 250–500 bp were excised from a 2% agarose gel and purified by a gel purification kit (Qiagen, Catalog # 28604) as per the manufacturer's instructions.