title: GSM5241879: SV_PIWI_IP_1; Drosophila melanogaster; ncRNA-Seq
library_construction_protocol: Samples prepared in Linkoping: Frozen samples were homogenized in Qiazol with 0.15 g 0.2 mm steel beads, Tissue Lyser 2 min 40 osc. RNA extraction was done using miRNeasy Micro kit (Qiagen, Venlo, the Netherlands) and performed according to the manufacturer instructions, RNA was eluted in 14 μl of water and stored at −70 °C until library preparation. Samples prepared in Freiburg: Dried RNA were shipped on dry ice and kept in -70 °C. Dried and washed once with 80% ethanol and dried, dissolved in 20 ul water, extraction was done using miRNeasy Micro kit (Qiagen, Venlo, the Netherlands) and performed according to the manufacturer instructions. RNA was eluted in 14 μl of water and stored at −70 °C until library preparation. Bioanalyzer confirmed the quality of the RNA. Library preparation was done with NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs, Ipswich, MA) according to the manufacturer instructions with the following minor customizations.  All testicle samples, but not sperm samples, were downscaled to half volume, using 3 μl of input RNA instead of 6 ul as recommended. In all steps the primers in the kit were diluted 1:3 prior to use. 2S rRNA was blocked by adding anti-sense oligos (5´-TAC AAC CCT CAA CCA TAT GTA GTC CAA GCA372 SpcC3 3´; 10 µM) and set to hybridized in the same step as NEBNext SR RT primer (pink). Amplification was made during 16 cycles and amplified libraries were cleaned using Agencourt AMPure XP (Beckman Coulter, Brea, CA) and size selected for 130 to 165 nt fragments on a pre-casted 6% polyacrylamide Novex TBE gel (Invitrogen, Waltham, MA). Gel extraction was done using Gel breaker tubes (IST Engineering, Milpitas, CA) in the buffer provided in the NEBNext kit. Disintegrated gels were incubated at 37 °C for 1 hour on a shaker, quickly frozen for 15 minutes at −80 °C, followed by another incubation for 1 hour. Any remaining gel debris was removed by Spin-X 0.45 μm centrifuge tubes (Corning Inc., Corning, NY) as recommended by the NEBnext protocol. The libraries were precipitated overnight at −80 °C by adding 1 μl of GlycoBlue (Invitrogen) as co-precipitant, 0.1 times the volume of Acetate 3M (pH 5.5), and 3 times the volume of 100% ethanol. Library 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 Illumina's default quality control.