Small RNA profiles of mouse sperm in response to male environmental exposures across age and genetic background
Small RNA profiles of mouse sperm in response to male environmental exposures across age and genetic background
Male mice were randomly assigned to one of three treatment groups: Control (CON), non-absorbable antibiotics (nABX), or low protein high sugar diet (LPHS). Young (3 weeks) and mature (12-13 months) mice in the CON group received water and standard rodent diet, nABX mice received antibiotics in their water (Neomycin, Bacitracin, Pimaricin) along with the standard diet, and LPHS mice were given a low-protein, high-sugar (LPHS) diet and water. Cauda epididymides from FVB and C57BL/6J control and treated mice were dissected, cleaned, and incubated in M2 media to allow spermatozoa to swim out. After 30 minutes at 37C and 5% CO2, remaining sperm was released by gently pinching tissues, followed by an additional 10-minute incubation. The sperm suspension was filtered, centrifuged, and resuspended in somatic cell lysis buffer on ice for 20 minutes to remove any somatic cell contamination. After further washes and centrifugation, sperm pellets were dried and stored at -80C. Sperm RNA was extracted using an optimized mirVana miRNA isolation kit. Briefly, spermatozoa were lysed in mirVana lysis buffer with a tissue homogenizer. The lysed solution underwent organic extraction and RNA isolation following the mirVana protocol, and including an Acid-Phenol: Chloroform step. RNA was precipitated, purified, washed, and eluted with nuclease-free water, and its concentrations were measured using the TapeStation, ranging from 1000 to 8000 pg/ÂµL. The RNA was stored at -80C. Libraries were constructed using the NextFlex Small RNA-Seq Kit v4 by PerkinElmer, with varying concentrations of starting material but consistent volumes across samples.