Rneasy kit protocol

The frozen PBMCs were thawed in bulk and RNA extracted as per the Qiagen RNeasy kit protocol. The quality of the extracted RNA was confirmed using Agilent Bioanalyzer and messenger RNA (mRNA) enrichment done. mRNA sequencing libraries for 200 bp short-inserts were prepared according to the manufacturer’s recommendations. RNA sequencing was done using the Hi-Seq Illumina platform using nine lanes of single-ended reads at a read length of 50 base-pairs (50 bp). RNA extraction, Library preparation, and sequencing were performed at the Beijing Genomics Institute (BGI), China.

RNA was isolated from peripheral whole blood stored in heparinized tubes at −80°C using a modi ed QIAGEN RNeasy Kit protocol (see Supplementary Methods 1.1). RNA templates were then utilized for complimentary DNA (cDNA) synthesis utilizing the iScript cDNA synthesis kit protocol (Bio-Rad).

In vitro and in vivo RNA extraction were carried out as described previously (7 (link), 9 (link)). For in vitro RNA extraction, cells were collected at different time points, centrifuged for 2 min at 10 K rpm at room temperature and RNA was extracted according to the manufacturer’s protocol (MasterPure Yeast RNA Purification Kit). For in vivo RNA extraction, mice were euthanized, ear tissue was harvested, and the tissue was placed into the ice-cold RNA-Later solution. Ear tissue was then transferred to the mortar and flash-frozen with liquid nitrogen. Using a pestle, the frozen was ground to a fine powder. The resulting powder was collected and 1 mL of ice-cold Trizol was added. The samples were placed on a rocker at RT for 15 min and then centrifuged at 10K at 4°C for 10 min. The cleared Trizol was collected into a 1.5 mL Eppendorf tube and 200 µL of RNase-free chloroform was added to each sample. The tubes were shaken vigorously for 15 s and kept at RT for 5 min followed by centrifuge at 12 K rpm at 4°C for 15 min. The cleared aqueous layer was then collected into a new 1.5 mL Eppendorf tube and RNA was further extracted following the Qiagen RNeasy kit protocol.

Testes were harvested and homogenized in Trizol reagent (Thermo Fisher) and stored at −80°C prior to processing. Total RNA was extracted from the aqueous phase, mixed with ethanol and purified using the RNeasy kit protocols and reagents (Qiagen). RNA was quantified using the Qubit RNA assay (Thermo Fisher) and 400–500 ng total RNA per sample was used in stranded mRNA-seq library preparation (KAPA Biosystems, KK8481) for Illumina sequencing. Libraries were pooled and sequenced with 2×150 cycles paired-end to an average depth of 19.4 million reads per sample on a HiSeq 4000 by Genewiz (South Plainfield, NJ, USA).

Total RNA was extracted using Qiagen RNeasy kit protocols (Qiagen, Hilden, Germany). The frozen punched samples were allowed to thaw to ambient temperature and QIAzol phase later was separated with 350 µl chloroform (15 min, 12,000 g, 4 °C). The upper aqueous phase was removed then mixed with 70% (v/v) ethanol to precipitate the total RNA, which was resuspended and applied to RNeasy columns in accordance with the manufacturer’s instruction. For RNAseq experiments, punched samples at QIAzol phase were pooled (5 per group), whereas individual samples were used in qRT-PCR (n = 6). The RNA concentration was measured using a Nanodrop spectrophotometer (ND-2000, Thermo Scientific, Wilmington, DE) and Qubit Fluorometer 2.0 (Life Technologies). The RNA samples were also analysed using 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA 95051) to obtain RNA integrity numbers (RIN numbers) as a measure of their quality (Schroeder et al., 2006 (link)). All RNA samples met the RIN quality criterion of >8.5.