title: GSM5640050: Degradome.mouse.testis.wt.Ribozero.adult.rep1; Mus musculus; OTHER
library_construction_protocol: Total RNAs were extracted using mirVana miRNA Isolation Kit (ThermoFisher, AM1560), and rRNAs were depleted from total RNAs with complementary DNA oligomers (IDT) and RNaseH (Invitrogen, Waltham, MA, USA) 1, Strand-specific RNA-seq: libraries were constructed following the TruSeq RNA sample preparation protocol. 2, Ribo-seq: Testes samples were lysed, RNase T1 & A treated and loaded on sucrose gradients, and 80S monosomes were recovered for library constrution. The 3'-adapter is TGGAATTCTCGGGTGCCAAGG. 3, Degradome: The RNAs were first oxidized at room temperature for 30 minutes with sodium periodate (Sigma, St. Louis, MO, USA) to block the 3´-ends from ligation, and were then size-selected to isolate RNA ≥ 200 nt (DNA Clean & Concentrator™-5, Zymo Research). 5´ adaptors were attached using T4 RNA ligase (Ambion) at 20°C for 3 hours. The ligated products were subjected to rRNA depletion with complementary DNA oligomers (IDT) and RNaseH (invitrogen). The rRNA depleted ligation products were reverse transcribed using a degenerate primer (5´-GCACCCGAGAATTCCANNNNNNNNC-3´). cDNA was amplified by PCR using KAPA HIFI Hotstart polymerase (Kapa Biosystems, Wilmington, MA, USA), and 250–350 nt dsDNA was isolated on 8% native PAGE gels. 4, Small RNAseq: Small RNA libraries were constructed and sequenced using oxidation to enrich for piRNAs by virtue of their 2´-O-methyl-modified 3´ termini. The oxidation procedure selects against in vitro-digested products with a 3´ phosphate. A 25-mer RNA with 2´-O-methyl-modified 3´ termini (5´-UUACAUAAGAUAUGAACGGAGCCCmG -3´) was used as a spike-in control. The 3'-adapter is TGGAATTCTCGGGTGCCAAGG.