title: Small RNA seq of zebrafish spermatozoa  from stressed adult male (batch4)
design: Adult male London Wildtype (LWT) zebrafish (aged 6-10 months) were housed individually in tanks on a recirculatory system with water heated to 26C and kept on a 12:12h light / dark cycle. Animals were fed brine shrimp or flake food twice daily. Adult males were subjected to a chronic stress constituting 20 minutes of alarm substance exposure, periodic net chasing, or bright light exposure for 12-14 days. Control males were handled in an identical fashion but not subjected to the stressors. Four to five individuals (in both control and stress groups) comprised one experimental batch. A total of six experimental batches were carried out.Spermatozoa were collected from euthanized adult males by disruption of dissected testes in ice-cold Hanks balanced salt solution (HBSS) to derive a testicular cell suspension, which was then centrifuged (800 xg for 40 min) on top of a 40/80 Percoll gradient to separate spermatozoa from larger cells. The sperm precipitate washed in ice cold HBSS, pelleted (1000 xg for 10 mins), re-suspended in Tri-reagent and stored at -80C until RNA extraction. Total RNA was extracted following the Tri-reagent protocol with the addition of an overnight precipitation step with glycogen in 2-propanol at -20C. Each sperm RNA sample comprised a pool of multiple individuals within the experimental batches such that each of six batches contributed one replicate pair (control and stress), for a total of 12 biological samples.Small RNA sequencing libraries were prepared using the SomaGenics (Santa Cruz) Real-Seq Biofluids kit following the manufacturers instructions, using a minimum of 2.5 ng of input total RNA. Twenty PCR cycles were performed to amplify the reverse transcription product. The quality of the prepared libraries was examined using the Agilent Hi-sensitivity DNA chip prior to the individual libraries being pooled to a concentration of 4nM for Illumina sequencing. Sequencing was carried out on an Illumina HiSeq 2500 using SBS v3 chemistry and single end 65 base reads.### PLEASE NOTE ###The supplied FASTQ files are raw data which still contain the adaptor sequence (TGGAATTCTCGGGTGCCAAGG).