GSM8619810: WT testis, 6-week-old, Ribo-seq, REP3; Mus musculus; OTHER
1.Tissue lysis; 2.Translation inhibition fixation; 3. Rnase digestion; 4. Ribosome digestion; 5. Ribosome profiling RNA extraction; 6. Ribosomal RNA removal cDNA libraries were prepared by Vertis Biotechnologie AG (Freising, Germany) using the adapter ligation protocol without fragmentation.   At first, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules.   First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3' adapter as primer.   The first strand cDNA was purified, and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA.   The resulting cDNA was PCR-amplified to about 10-20 ng/μl using a high-fidelity DNA polymerase.   The DNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina.