GSM9158892: Pool_Perfect_2; Mus musculus; OTHER
In vitro slicing: The samples containing the in vitro sliced RNAs were phenol-chloroform extracted and precipitated with ethanol. piRNA sequencing: To extract MIWI-bound piRNAs from adult mouse testes, eluted StrepII-MIWI was subjected to a proteinase K treatment. Briefly, two micrograms of StrepII-MIWI were digested with 10 μg of Proteinase K in Proteinase K buffer by incubation at 42°C for 20 min (with shaking at 400 rpm). The reactions were immediately phenol-chloroform extracted as standard and precipitated overnight in ethanol. After centrifugation of the samples to precipitate the piRNAs, the dry pellets were resuspended in 20 μL RNase-free water. Gene expression analysis: Around 500,000 cells from 1n (spermatids) and 4n (spermatocytes) sorted samples from testicular single cell suspensions were used as input for RNA extraction. RNA was extracted using the Allprep DNA/RNA Micro Kit (Qiagen). The extraction was carried out according to the product's manual. Elution was performed with 15 uL DEPC water. In vitro slicing: The precipitated RNA samples were used as input for small RNA library preparation (6 μl of the precipitated RNA following phenol-chloroform extraction), about 80 ng starting material. Libraries were prepared using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB) and sequenced with the Illumina HiSeq 2000 platform (EMBL GeneCore facility, Heidelberg) using the P50 paired-end option. piRNA sequencing: Libraries were prepared using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB) and sequenced with the Illumina HiSeq 2000 platform (EMBL GeneCore facility, Heidelberg) using the P50 paired-end option. Gene expression analysis: The Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit was used for the library preparation with 100 ng of total RNA as input. The libraries were sequenced on an Illumina NovaSeq 6000 sequencer for paired-end 100 reads.