title: GSM5733302: dme.WT.tes.rep3; Drosophila melanogaster; RNA-Seq
library_construction_protocol: 1) For testis samples, adult flies 3-5 days after eclosion were allowed to mate and then testes were dissected in drops of 1× phosphate-buffered saline (PBS) with two pairs of Dumont #5 forceps. For the extraction of total RNA, 20-25 pairs of testes were pooled as a sample. RNeasy Micro Kit (QIAGEN, German) was used for RNA extraction following the manufacturer's instructions. 2) For single-cyst samples, testes from mated male flies 2-3 days after eclosion were dissected. Two tungsten micro-needles (World Precision Instruments, USA) were used to tear the muscle sheath layer, resulting in germ cell cysts spilling into a drop of 1× phosphate-buffered saline (PBS). The cysts were viewed using an inverted microscope (Leica DMI4000 microscope), and were picked using a glass capillary (BJ-40, Beijing Zhengtian Yi Science and Trade Co. LTD) pulled by a Sutter instrument P-30 with the following parameters: heat#1 = 930; pull = 909. The cysts were staged using the criteria as follows: spermatogonia (SG), 16-cell cysts with cells of <10 μm in diameter for D. melanogaster and < 8 μm in diameter for D. simulans; late primary spermatocytes (pSC), 16-cell cysts with cells of 22-25 μm in diameter; round spermatids (rST), cysts with much more than 32 round or nearly round cells of 14-16 μm in diameter; and elongating spermatids (eST), bundles of 250-350 μm in length. The staged single cyst was transferred into PCR tube by pushing the syringe which was connected with the glass capillary by an infusion tube. Cell lysis, reverse transcription, PCR preamplification and purification of PCR products were conducted using Discover-sc™ WTA Kit V2 (Vazyme Biotech, China) according to the user's guides. 1) For testis samples, RNA libraries were prepared for sequencing using standard Illumina protocols. 2) For single-cyst samples, RNA-seq libraries were constructed using TruePrep™ DNA Library Prep Kit V2 for Illumina® (Vazyme Biotech, China) and an input of 1 ng amplified cDNAs.