Megashortscript t7 high yield transcription kit

BE3 and ABE7.10 plasmids were purchased from Addgene (#73021 and #102919). After linearization with NotI, the plasmid underwent in vitro transcription using mMESSEAGE mMACHINE T7 kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and was purified by LiCl precipitation. sgRNAs were amplified and transcribed in vitro using MEGAshortscript T7 High Yield Transcription Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions. Primers are listed in S2 Table. After transcription, sgRNAs were purified by ethanol precipitation.

To detect fgf9 and rspo1 expression in gonadal tissue, two pairs of primers were designed to obtain a riboprobe (Table 1). A 468 bp cDNA fragment was amplified from the fgf9 ORF domain, and a 533 bp cDNA fragment was amplified from rspo1. Primers were designed to introduce the T7 promoter sequence in the 5′- or 3′- side of the amplicons that will later be used as riboprobes, and PCR products were purified by a QIAquick Gel Extraction Kit (QIAGEN, Cat# 28706). To obtain probes, transcription was performed using the MEGAshortscript T7 High Yield Transcription Kit (Invitrogen, AM1354) according the manufacturer’s instructions In situ hybridization was performed following the method described in previous study (Hu et al., 2016 (link)). Sections were deparaffinized, hydrated and treated with proteinase K and then hybridized using sense or antisense DIG-labeled RNA probe at 70°C for 12 h. Hybridization signals were then detected with anti-DIG (Roche, 11093274910) conjugated with POD. DAB (Beyotime, P0202) was used as substrate for POD (Hu et al., 2016 (link)).

Biotin-14-CTP (Thermo Fisher Scientific, Cat# 19519016) labeled RNA was in vitro transcribed using MEGAshortscript™ T7 High Yield Transcription Kit (Thermo Fisher Scientific, Cat# AM1354) according to the manufacturer's instruction. After purification with RNeasy Plus Mini Kit (Qiagen, Cat# 74136), 2 µg biotinylated RNAs was incubated with 20 µL pre-washed Dynabeads™ M-270 Streptavidin (Thermo Fisher Scientific, Cat# 65305) in 1 mL buffer (20 mM Tris–HCl (pH7.5), 100 mM KCl, 5 mM MgCl₂, and 0.5% NP-40), and incubated at 4 °C for 1 h with gentle agitation. 450 µg mIEC cytoplasmic protein lysate was added to the washed RNA-probe coupled dynabeads, and incubated in 1 × TENT buffer (10 mM Tris–HCl (pH8.0), 1 mM EDTA (pH 8.0), 250 mM NaCl, and 0.5% Triton X-100) with SIGMAFAST™ protease inhibitor cocktails and RNaseOUT™ Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific, Cat# 10777019) at 4 °C for 8 h. After washed with 1 × TENT buffer, the proteins interacted with mouse lgr5 probes were eluted using 40 µL SDS buffer and detected by immunoblot assay. Biotinylated Gapdh RNA was used as a pull-down control. All RNA-probes are listed in Table S3.

To assess plastin-2 expression in gonadal cells, two pairs of primers were designed (Additional file 1: Table S5). A 433 bp cDNA fragment was amplified from the plastin-2 Open Reading Frame (ORF) domain. Primers were used to amplify the T7 promoter sequence synthetic probe, and PCR products were purified using a QIAquick Gel Extraction Kit (QIAGEN). Transcription was performed using the MEGAshortscript T7 High Yield Transcription Kit (Thermo Fisher Scientific) to obtain probes. In situ hybridization was performed as described in a previous study [36 (link)] using anti-digoxigenin-AP Fab fragments (Roche) as the antibody, while BCIP/NBT (Beyotime) was used to detect the positive signal.

To assess the expression of MSTRG.12998 and MSTRG.38036 transcripts and their target genes in gonadal cells, primers were designed according to the sequences to amplify the T7 promoter sequence with a synthetic probe (Additional file 1: Table S1). PCR products were purified by a QIAquick Gel Extraction Kit (QIAGEN, Germany). The MEGAshortscript T7 High Yield Transcription Kit (ThermoFisher Scientific, USA) and DIG RNA labelling mix (Roche, Switzerland) were used to obtain probes. According to a previous description [25 (link)], anti-digoxigenin-AP Fab fragments (Roche, Switzerland) were used as the antibody, and BCIP/NBT (Beyotime, China) was used to detect the positive signal.