GSM6923078: Mael+/- RNA, rep2; Mus musculus; RNA-Seq
Freshly dissected testes were collected in ice-cold PBS supplemented with 200 µg/ml cycloheximide and homogenized in Lysis buffer (McGlincy and Ingolia, 2017). Lysates were clarified by centrifugation, supernatants were recovered and the RNA concentration was determined using a Qubit Fluorometer (Thermo Fisher Scientific). For total RNA libraries, aliquots of testicular extracts containing 2.5 µg of RNA were processed for RNA purification and libraries were generated using the TruSeq Stranded Total RNA kit (Illumina), with Ribo-Zero rRNA depletion. For ribofootprint libraries, aliquots of testicular extracts containing 20 µg of RNA were incubated with 5U MNase (Takara Bio) to generate monosomes. Ribosomes were then processed for footprints purification and libraries generation as described in McGlincy and Ingolia, 2017, with the following modifications: our linker sequence is: /5rApp/NNN NNA TCG AGA TCG GAA GAG CAC ACG TCT GAA CTC/3ddC/ (Santos et al., 2019); footprints were reverse transcribed using the following RT primer: /5Phos/AGA TCG GAA GAG CGT CGT GTA GGG AAA GAG/iSp18/CTG GAG TTC AGA CGT GTG (Santos et al., 2019).