Preparing Semen Samples for Separation 1. Determine the number of tubes needed for isolations based on the volume used for DNA/RNA. You will use ¼ of the total semen volume for DNA isolation and the remaining ¾ for RNA isolations. a. Up to 1 ml of semen can be used per conical tube b. Example: 4 mls semen i. 1 ml is for DNA → therefore 1 conical tube is required for DNA analysis prep ii. 3 mls for RNA → therefore 3 conical tubes are required for RNA analysis prep 2. Label tubes with sample number and either “RNA” or “DNA” Sperm Separation: Percolling: 1. When preparing Percoll and 2x PBS, do under sterile conditions to keep the Percoll sterile. a. Add up to 1 ml 2x PBS and 1 ml Percoll to a 15 ml conical tube. 2. Gently pipette up to 1 ml semen sample on top. 3. Spin samples at 1200 rpm for 40 min → will see visible pellet at the bottom 4. Remove supernatant until the interface layer right before the pellet in bleach. Sperm Separation: Washing: 1. Combine sperm pellets and remaining supernatant from DNA and RNA tubes, respectively, to reduce the number of tubes you are working with. 2. Add 1x PBS to a total volume of 10 ml. 3. Spin samples at 2000 rpm for 7 minutes and remove as much supernatant as you can without disrupting sperm. 4. Repeat washes 2x. 5. Resuspend pellet in 1 ml 1x PBS. 6. Add suspensions to properly labeled tubes for DNA and RNA. 7. Spin DNA tube at max speed to pellet the cells. 8. Remove supernatant, flash freeze pellets in liquid nitrogen, and store in -80 freezer. 9. Continue with RNA isolation protocol.
Extracted molecule
genomic DNA
Extraction protocol
DNA was isolated from the sperm of the 21 men using a modified protocol in which sperm pellets were lysed for 16 hours in a solution containing Tris (Fisher Scientific, Pittsburgh, PA, USA), DTT (Promega Corporation, Madison, WI, USA), NaCl (EMD Chemicals, Inc., the North American Affiliate of Merck KGaA, Darmstadt, Germany), EDTA (Fisher Scientific, Pittsburgh, PA, USA), SDS (Fisher Scientific, Pittsburgh, PA, USA), Proteinase K (Promega Corporation, Madison, WI, USA), and beta-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) [32]. The DNA was then extracted using phenol/chloroform (Sigma-Aldrich, St. Louis, MO, USA), ethanol precipitated, and bisulfite modified using the EZ DNA Methylation kit (Zymo Research Corporation, Orange, CA, USA).
Label
Cy5 and Cy3
Label protocol
Standard Illumina Protocol
Hybridization protocol
bisulphite converted DNA was amplified, fragmented and hybridised to Illumina Infinium Human Methylation27 Beadchip using standard Illumina protocol
Scan protocol
Arrays were imaged using BeadArray Reader using standard recommended Illumina scanner setting
