Wt_b_Testicle_part_2
Frozen samples were homogenized in Qiazol with Tissue Lyser 2 min 40 osc, using a 0.15 g 0.2 mm steel beads,. RNA extraction was done using miRNeasy Micro kit (Qiagen, Venlo, the Netherlands). RNA was eluted in 14 l of water and stored at 70 C. Libraries were prepared with NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs, Ipswich, MA) with minor customizations. All testicle samples, but not sperm samples, used 3 ul of input RNA instead of 6 ul. Primers were diluted 1:3. 2S rRNA was blocked with anti-sense oligos (5-TAC AAC CCT CAA CCA TAT GTA GTC CAA GCA372 SpcC3 3; 10 M) set to hybridized in the step when NEBNext SR RT primer (pink) were added. Libraries were amplified for 16 cycles and cleaned using Agencourt AMPure XP (Beckman Coulter, Brea, CA). Size selection was applied for 130 to 165 nt fragments on a pre-casted 6% polyacrylamide Novex TBE gel (Invitrogen, Waltham, MA). Gel breaker tubes were used for gel extractions (IST Engineering, Milpitas, CA) in the NEBNext kit buffer. Gels were incubated at 37 C for 1 hour on a shaker, snap frozen for 15 minutes at 80 C, followed by another incubation for 1 hour. Remaining gel debris was removed by Spin-X 0.45 m centrifuge tubes (Corning Inc., Corning, NY).  Size-selected libraries were precipitated overnight at 80 C by adding 1 l of GlycoBlue (Invitrogen) as co-precipitant, 0.1 times the volume of Acetate 3M (pH 5.5), and 3x volume of 100% ethanol. Concentrations were estimated using QuantiFluor ONE ds DNAsystem on a Quantus fluorometer (Promega, Madison, WI). Pooled libraries were sequenced on NextSeq 500 with NextSeq 500/550 High Output Kit version 2, 75 cycles (Illumina, San Diego, CA). All pooled libraries passed Illuminas default quality control.