Rattus norvegicus Riboseq

Ribosome profiling was done from SD rat cortical synaptoneurosomes (Ingolia N. T. et al., 2012) with few modifications. SNS were stimulated DHPG/NMDA for 5min, lysed in cycloheximide containing lysis buffer and pelleted at 20,000g for 20min. 800µl was used to treat with RNase I ((100 U/µl; Invitrogen, cat. no. AM2294) for 45min, the rest of the lysate was kept for total RNA sequencing. RNase treated lysate was then added on 1M sucrose solution and spun at 70,000 rpm for 4h in TLA 100.3 rotor. The ribosome pellet obtained was then dissolved in TRIzol for RNA extraction. RNA obtained was then run on 15% urea-PAGE for 2h at 200V. Bands between 34-25 nt were cut, and RNA was eluted out from the gel using 0.3M NaCl. Pelleted RNA was then dephosphorylated at the 3' end with PNK and phosphorylated at the 5' end with PNK and 1mM ATP for 1h at 37°C. Library was prepared using illumina TruSeq Small RNA sample preparation kit. Product size of approx. 180bp was used to load on the Nextseq illumina sequencer to obtain 120 million reads 75 SE, per sample.