Qpcr master mix

The GGBC at the University of Georgia conducted all library preparation and sequencing. The KAPA Stranded mRNA-Seq kit was used for the construction of NGS stranded RNA library for each replicate (KK8421, KAPA Biosystems, Wilmington, MA, USA). All libraries were pooled together by qPCR using the Roche LightCycler 480 II (product no. 05015278001, Roche Molecular Systems, Inc., Pleasanton, CA, USA). For this step, the KAPA Library Quantification kit (Illumina) with qPCR Master Mix optimized for LightCycler 480 was used (KK4854, KAPA Biosystems, Wilmington, MA). Next, the pooled library underwent pre-sequencing quality control. The DNA concentration of the pooled library was quantified using the Qubit HS dsDNA assay (catalog no. Q32854, ThermoFisher Scientific, Waltham, MA, USA). The Fragment Analyzer Automated CE System (Advanced Analytical Technologies, Ankeny, IA, USA) was used to visual the size distribution of the library. Then, qPCR was performed using the same kit and PCR products were quantified as described above. The pooled libraries were sequenced on an Illumina NextSeq 500 for 150 cycles with a PE75 high output flow cell.

Total RNA was extracted from mouse forebrain tissues at P8 using the RNeasy Plus Mini Kit (Qiagen, 74104, Hilden, Germany). The RNA concentration and quality were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). One microgram of total RNA was reverse transcribed to cDNA using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, RR047A, Kusatsu, Japan). Real-time qPCR was performed on a StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer’s instructions. The reaction mixture comprised Roche’s qPCR Master Mix (Roche, 4913850001, Basel, Switzerland) and specific primers designed for the target genes. The primer sequences were listed in the Supporting Information Table S1. The relative expression levels of target genes were normalized to the expression of GAPDH and calculated using the 2^−ΔΔCt method.

The total RNA was isolated as described above. The total RNA was quantified using a Nanodrop 3300 (ThermoScientific, Waltham, MA, USA) [51 (link)]. Two micrograms of the total RNA were used to make the first strand of complementary DNA (cDNA; in 20 µL) using an RT2 First Strand Kit (Cat. No: AT311-192 03, Transgenic Biotech, Beijing, China) following the manufacturer’s instructions. The generated first-strand cDNAs (20 µL) were diluted to 120 µL with double-deionized water (ddH₂O). Then, 1 µL was used for one PCR reaction (in a 96-well plate). Each PCR reaction (12 µL) contained 6 µL of qPCR Master Mix (Roche, Mannheim, Germany), 1 µL of diluted first-strand cDNA, 0.6 µL primers (10 µM), and 4.4 µL of ddH₂O. The primers for the qPCR analysis were synthesized by Invitrogen and are presented in Table 1. The qPCR was conducted with the Roche LightCycler^@ 480 (Roche, Germany) with the following program: Step 1: 95 °C, 10 min; Step 2: 40 cycles of 95 °C, 15 s; 60 °C, 1 min; Step 3: dissociation curve; Step 4: cool down. Three or more independent experimental samples were analyzed.

Total RNA was extracted from testicular tissues using an RNA extraction kit (Sparkjade Biotechnology Co., LTD, Shandong, China). Then, 2 μg of RNA was reverse transcribed using HiScript Ⅱ1 s Strand cDNA Synthesis Kit (+gDNA wiper) obtained from Vazyme Biotech Co., Ltd. Briefly, 2 μg of total RNA was used to make the first strand of complementary DNA (cDNA; in 20 μL) using an RT2 First Strand Kit (Cat. No: AT311-03, Transgen Biotech, Beijing, China) following the manufacturer’s instructions. The generated first-strand cDNAs (20 μL) were diluted to 150 μL with double-deionized water (ddH₂O). Then, 1 μL was used for one PCR reaction (in a 96-well plate). Each PCR reaction (12 μL) contained 6 μL of qPCR Master Mix (Roche, Basel, Switzerland), 1 μL of diluted first strand cDNA, 0.6 μL primers (10 mM), and 4.4 μL of ddH2O. The primers for qPCR analysis were synthesized by Tsingke. qPCR was conducted by using a Roche LightCycler 480 (Roche, Basel, Switzerland) with the following program: step 1: 95 °C, 10 min; step 2: 40 cycles of 95 °C, 15 s; 60 °C, 1 min; step 3: dissociation curve; step 4: cool down; n = 3/group. Additionally, the relative abundance of mRNA was calculated and normalized to mean β-actin mRNA levels. The primer sequences used in this study are presented in Table 1.