title: Total RNA-seq on round spermatids from FVB/NCrl-Trim66em2(gfp)Emr mice
design: Total RNA-seq on round spermatids from FVB/NCrl-Trim66em2(gfp)Emr mice
library_construction_protocol: The genotype for KO is homozygous for the mutation  "FVB/NCrl-Trim66em2(gfp)Emr". The homozygous mutants were produced by intercrossing heterozygous for the allele FVB/NCrl-Trim66em2(gfp)Emr. The allele FVB/NCrl-Trim66em2(gfp)Emr was created at EMBL. Spermatids were isolated from testes by fluorescent activated cell sorting (FACS) using a BD FACS ariaII (BD Biosciences). The material was sourced from both testes for each mouse and was run until the whole material was sorted. The dissected tissue was incubated in enhanced Krebs-Ringer's buffer (120 mM NaCl, 4.8 mM KCl, 25.2 mM NaHCO3, 1.2 mM KH2PO3, 1.2 mM Mg2SO4, 1.3 mM CaCl2, 11mM glucose, 1X Non-essential AA (Gibco), Pen/Strep 1X (Gibco)). De-encapsulated testes were digested for 5 min in 0.5 mg/ml collagenase (Clostridium histolyticum, Sigma), then treated for 10 min with Trypsin (Acetylated from bovine pancreas, type V-S) at 32 °C. The staining was done in EKRB buffer with 10% FBS, with 10ug/ml Hoechst 33342 for 30 min and 10 min with 2 µg/ml propidium iodide at 32 °C. The cells were sorted in a sorting buffer (10% FBS enhanced Krebs-Ringer's buffer). The sorted spermatid fractions were collected in Monarch DNA/RNA protection reagent (NEB) and flash frozen in liquid nitrogen before the RNA extraction. The animals were sacrificed by cervical dislocation. The testis were extracted from the abdominal cavity pulling the fat pads surroundings tissue. The epidyndymis was surgically detached and excess of fat removed in suspension of 1x PBS. The tissue was washed once in 1x PBS before the de-encapsulation of the testis tubules. After removal of the tunica the testis were placed in EKRB buffer before the enzymatic tissue dissociation. Total RNA was extracted from collected spermatids fractions with Monarch RNA extraction kit (NEB) according to manufacturers' protocol for RNA extraction from tissue or leukocytes, with proteinase K incubation time optimised for homogenized tissues. The optional step with DNAseI treatment was performed plus an additional steps with TurboDNase to eliminate all traces of genomic DNA (Invitrogen). Total RNA was then purified with Monarch RNA Cleanup Kit (NEB) and quantified using Qubit Fluorimetric Quantification (Thermo Scientific). The integrity of the RNA was determined using the TapeStation High Sensitivity RNA kit (Agilent Technologies). 10 ng of total RNA has been used to prepare ribo-depleted RNA-seq libraries with these kits: NEBNext rRNA Depletion Kit (Human/Mouse/Rat) and NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®. We used 17 PCR cycles to obtain enough material for sequencing.