GSM6568753: DEP_PSD95_pd_rep_1 [Ribo-Seq]; Mus musculus; OTHER
RNA from inputs and streptavidin pulldowns (from beads directly) was extracted using the TRIzol protocol.   [Ribo-Seq] RNA from inputs and streptavidin pulldowns was resolved by electrophoresis through a denaturing gel to isolate ribosome footprints. The expected size range fragments were extracted, and 3' ends of these RNA fragments were treated with T4 polynucleotide ligase to ligate a pre-adenylated barcoded DNA linker to be able to pool samples. After treatment with deadenylase and RecJ, inputs and pulldowns were pooled, purified with Zymo columns, and were depleted of ribosome RNA (rRNA) using Ribo-Zero. Following rRNA removal, RNA was used as template for reverse transcription, using an oligo specifically designed to be able to sequence from the 5'end of the ribosome protected fragment. The single-stranded cDNA is then circularized using an optimized CircLigase after another gel purification step of full-length products. A double stranded DNA library is generated using the circularized cDNA fragments that are amplified with the Kapa enzyme and a fixed primer combined with an Illumina index encoding primer. The resulting products are gel purified from a non-denaturing gel and quantified on a bioanalyzer. Pooled libraries were then sequenced on the Illumina Novaseq 6000 sequencer.  [Ribo-Seq]