Taqman small rna probes

Taqman cDNA synthesis was performed as previously described (Weiser et al., 2017 (link)). Briefly, for quantification of piRNA levels, TaqMan small RNA probes were designed and synthesized by Applied Biosystems. All piRNA species assessed by qPCR were normalized to U18 small nucleolar RNA. Fifty nanograms of total RNA was used for cDNA synthesis. cDNA was synthesized by Multiscribe Reverse Transcriptase (Applied Biosystems) using the Eppendorf Mastercycler Pro S6325 (Eppendorf). Detection of small RNAs was performed using the TaqMan Universal PCR Master Mix and No AmpErase UNG (Applied Biosystems). For quantification of mRNA levels, cDNA was made using 500 ng of total RNA using Multiscribe Reverse Transcriptase (Applied Biosystems). For quantification of snRNA levels, cDNA was made using 250 ng of total RNA using SuperScript III Reverse Transcriptase (ThermoFisher). Assays for mRNA and snRNA levels were performed with Absolute Blue SYBR Green (ThermoFisher) and normalized to eft-2 using CFX63 Real Time System Thermocyclers (Bio-Rad). All qPCR primers used are listed in Supplementary file 4.