GSM8207918: Bulk testis, Paf1L transheterozygous mutants, rep1; Drosophila melanogaster; RNA-Seq
For bulk testis RNA-seq samples: RNA was isolated from testis tissues of 1-3 days old transheterozygous tPAF mutants and control (w1118) flies. Tissues were dissected in cold 1x PBS on ice. Samples were homogenized and lysed in 1mL TRIzol and extracted according to standard procedure.  For RNA-seq of sorted spermatocytes: For spermatocyte isolation, testis tissues were dissected from 1-3 days old knock-in LAP-Cdc73L flies (control or in combination with Paf1L or Leo1L mutant alleles) into cold Schneider's medium on ice. Following, the medium was removed and the tissue was homogenized with a plastic pestle and lysis buffer (560 µL PBS, 70 µl 1x trypsin, 70 µl 2% collagenase) was added. Samples were rotated at room temperature for 30 minutes followed by careful pipetting with a p1000 tip. The cell suspension was then drawn through a 40 µm mesh cell strainer into 500 µL Schneider's medium. 100 µL additional Schneider's medium was drawn through the cell strainer to collect the remaining cells. Samples were centrifuged at 1200 rpm for 7 minutes at 4°C and the supernatant was removed. Then, the cells were resuspended in 200 µL Schneider's medium, and DNA was stained with bisbenzimide Hoechst 33342 (Sigma). GFP-expressing cells were then isolated by fluorescence-activated cell sorting (FACS) on a FACSAria III machine (BD Biosciences, FACS Core Facility at Aarhus University, www.facs.au.dk). Flowjo software (Treestar) was used for data analysis. The preparation and FACS sorting of control and mutant spermatocytes were performed in three independent experiments. To this end, we isolated control and mutant spermatocytes with a purity of at least 85% by FACS sorting and characterized their transcriptome by RNA-seq. Wildtype testes were used to clearly identify the GFP-expressing spermatocyte population during flow cytometry. RNA isolation and library preparation were done in the same manner as for whole testis. Library construction for bulk testis RNA-seq: Genomic DNA was removed from the purified RNA samples using RNA Clean & Concentrator™-5 columns with on-column DNase digest (Zymo Research, R1014). For rRNA-depleted samples, rRNA was removed with RNase H using a modified version of the original protocol in (Morlan et al. 2012) as described in (ElMaghraby et al. 2019). Briefly, rRNA depletion was performed using a mixture of antisense oligos complementary to the mature Drosophila melanogaster rRNA transcripts. The oligo pool was mixed with 1 μg DNAse-free total RNA in RNase H Buffer (20 mM Tris-HCl pH 8, 100 mM NaCl) and annealed over a temperature gradient from 95°C to 45°C. The RNA-DNA hybrids were then digested with thermostable RNase H (NEB, M0523S) at 45°C for 1 hour. Next, RNA was purified and the DNA oligos were removed from samples using RNA Clean & Concentrator™-5 columns with on-column DNase digest (Zymo Research, R1014) according to the manufacturer's instructions. rRNA depletion was verified by qPCR. Library preparation was done with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, E7770L), following the recommended kit protocol. Amplified libraries were purified by SPRI beads and library quality was assessed with Bioanalyzer High Sensitivity DNA Analysis (Agilent Technologies, 5067-4626). Samples were sequenced on a NovaSeq 6000 sequencing system  (Illumina).  Library construction for sorted spermatocyte RNA-seq: Sorted spermatocyte RNA-seq libraries were prepared for each sample with and without rRNA depletion to minimize technical biases from working with small sample amounts. For the analyses, both library variants were pooled and analyzed together (see also the section on Analysis of RNA-seq data). Otherwise, the library preparation was performed as described above for whole testis RNA-seq.