Dnase 1 kit

Total RNA was extracted from whole testes or enriched cells using RNAzol RT (Molecular Research Center. Inc, RN 190) following the manufacturer’s instructions. After removing the residual genomic DNA with the DNase I Kit (Promega, M6101), 500 ng of total RNA was reverse-transcribed into cDNAs using the PrimeScript RT Reagent Kit (TaKaRa, RR037A) according to the manufacturer’s protocol. qRT-PCR was performed using an Eva Green 2× qPCR MasterMix-No Dye kit (Abm, MasterMix-S) on a LightCycler 480 instrument (Roche). Relative gene expression was analyzed based on the 2^−ΔΔCt method with β-actin as internal controls. At least three independent experiments were analyzed. All primers were listed in Supplementary file 1.

Total RNA was extracted from whole testes using Trizol reagent (Invitrogen, 15596-026) following the manufacturer's instructions. After removing the residual genomic DNA with the DNase I Kit (Promega, M6101), 500 ng of total RNA was reverse-transcribed into cDNAs using the PrimeScript RT Reagent Kit (TaKaRa, RR037A) according to the manufacturer's protocol. Real-time RT-PCR was performed using a SYBR Premix Ex Taq kit (TaKaRa, DRR420A) on a LightCycler 480 instrument (Roche). Relative gene expression was analysed based on the 2^−ΔΔCt method with Hprt and Gapdh as internal controls.
Primers used to determine the amount of Tuba3a, Tuba3b and Tubb4b pre-mRNA were targeted to the 3′ end of exon3 and the neighbouring 5′ end of intron 3. Primers used to determine the abundance of mature mRNAs of Tuba3a, Tuba3b and Tubb4b were designed to span an exon-exon junction, with Tuba3a-mRNA primers annealed to the 3′ end of exon4 and 5′end of exon5, Tuba3b-mRNA primers annealed to the 3′ end of exon1 and 5′end of exon2 and Tubb4b-mRNA primers annealed to the 3′ end of exon2 and 5′end of exon3.
The information of all primers was listed in Supplementary Table 5.