title: GSM6928089: Old_input_rep2; Mus musculus; RIP-Seq
library_construction_protocol: To prepare samples for the IP, the appropriate volume (80 µl per sample) of Dynabeads™ Protein G (Invitrogen, 10004D) was washed two times in 250 µl homogenization buffer (HB: 50 mM Tris–HCl pH 7.4, 100 mM KCl, 12 mM MgCl2 and 1% v/v NP-40) on a rotor at 4 °C for 5 min per wash. Two additional washes were performed with 250 µl supplemented HB (sHB: HB supplemented with 1 mM DTT (sigma 646563), 1× HALT protease inhibitor (Fisher P178441), 200 units/ml RNaseOUT, 200 units/ml RNasin (Promega N2115), 100 µg/ml CHX) on a rotor at 4 °C for 5 min per wash. Following the final wash, the beads were eluted back to the original volume with sHB. Oocyte samples were thawed, pooled to yield a total of 200 oocytes per replicate, and 300 µl sHB was added to each pooled sample. To lyse the cells, samples were vortexed for 30 s and the homogenates were centrifuged for 15 min at 20817 g at 4 °C. Ovaries were homogenized in 300 µl sHB and centrifuged likewise. To pre-clear the samples, the supernatants were transferred to new tubes, 20 µl of washed beads were added to each supernatant, and samples were incubated on a rotor at 4 ◦C for 30 min. A magnetic rack was used to remove the beads, and 30 µl (10 %) of each pre-cleared lysate was collected and added to 250 µl RLT buffer (Qiagen, 74034) supplemented with 1 mM DTT to serve as the input samples. The input samples were frozen and kept at −80 ◦C until RNA extraction. 4 µl (4 µg) anti-HA.11 epitope tag antibody (1/75, Invitrogen, 26183) was added to each of the extracts and all samples were incubated on a rotor at 4 °C for 4 hours. Thirty microliters of washed beads was added to the samples and incubated overnight on a rotor at 4 °C. The beads (now bound by HA-tagged ribosomes and the associated mRNAs) were washed four times in 750 µl urea wash buffer (uWB: 50 mM Tris–HCl pH 7.4, 150 mM KCl, 12 mM MgCl2, 1 % v/v NP-40, 1× protease inhibitors, 1 mM DTT, 40 U RNaseOUT, 40 U RNasin, 100 µg/ml cycloheximide, and 1 M urea) on a rotor at 4 °C for 10 min per wash. The bead slurry was then transferred to fresh tubes, pelleted via magnetic rack, and the uWB removed. To strip the mRNA complex off the beads, 250 µl RLT buffer supplemented with DTT was added to each sample and samples were vortexed for 30 s. Finally, Beads were pelleted and removed before RNA extraction was performed using the Rneasy Plus Micro Kit (Qiagen, 74034). Samples were eluted in 14 µl of RNase-free water and used downstream for RNA-Seq or RT-qPCR analysis. RiboTag RNA samples were sent to the Gladstone Institutes Genomics Core for quality control using Bioanalyzer (Agilent) and cDNA library preparation. cDNA was prepared with the Ovation RNA-Seq System V2 Kit (NuGEN, 7102), which uses a proprietary combination of enzymes and primers to preferentially prime non-rRNA sequences. First-strand cDNA synthesis is achieved with random hexamer primers and chimeric poly(T) primers, which bind to the region spanning the end of the 3'-UTR and the start of the poly(A) tail. Because stable mRNAs have at least 20 adenosine residues, use of these chimeric poly(T) primers allows for preferential priming of adenylated messages (mRNAs) with minimal bias for messages with longer poly(A) tails. RNA-Seq libraries were constructed using the Ovation Ultralow System V2 (NuGEN, 0344) and analyzed by Bioanalyzer, quantified by qPCR (Kapa Biosystems, KR0405), and sequenced using the HiSeq 4000 system (Illumina).