Recombinant ribonuclease inhibitor

Mice treated with either VPA or saline for 7 days were anesthetized deeply with isoflurane, and whole brains were rapidly removed and frozen on PND15. Using a cryostat (CM1850, Leica, Wetzlar, Germany), frozen brains were mounted on the specimen discs and were cut coronally to expose the caudal end of the RVLM. Bilateral RVLM tissues were removed using an 18-gauge needle according to the mouse brain atlas of Franklin and Paxinos [61 ] and stored at −80 °C. Total RNA was extracted using the RNeasy mini kit (QIAGEN, Hilden, Germany). The RNA quality and concentration were assayed using a NanoDrop 2000 device (Thermo Fisher Scientific, Waltham, MA, USA). Purified total RNA was reverse-transcribed into cDNA by incubating with a recombinant ribonuclease inhibitor (TaKaRa Bio, Tokyo, Japan), 5× prime script buffer (TaKaRa Bio), 5× reverse transcriptase M-MLV Buffer (TaKaRa Bio), and random primer (Invitrogen, Carlsbad, CA, USA). A quantitative reverse transcription PCR (RT-qPCR) assay was performed with SYBR Premix Ex Taq II (TaKaRa Bio), and data analysis was carried out with the use of CFX96-Real-Time System (Bio-Rad, Hercules, CA, USA). For the quantification of mRNA, the target expression level was normalized to the Gapdh (glyceraldehyde-3-phosphate dehydrogenase) mRNA level. The primer sequences used for RT-qPCR are shown in Table A2.

Sperm was isolated in TC-100 Insect Medium (Sigma; #T3160) essentially as described (Öst et al, 2014 (link)). From each diet, sperm from 15 flies were dissected and pooled in 1:10 dilution of RNAse inhibitor (Recombinant ribonuclease inhibitor 5000 U; Cat. 2313A Takara); samples were flash-frozen on dry ice and later stored at −80°C. For sperm collection, five samples of each diet were prepared.
In the repeated experiment of three sugar diets for verification of results, six samples per diet were prepared, in total 18 samples.

The processing site was mapped by reverse transcription of the processed transcript using an oligonucleotide (showed in Additional file 1: Table S2) complementary to positions 16–25 bp of the mcherry, a sequencing ladder starting at the 5′end of the transcript was run in parallel. In the case of the full-length transcript, the second fragment originating from this cleavage site has a predicted length about 150 bp corresponding to the sequences between the cleavage site and the 3′end of the transcript.
To map the 5′-terminal nucleotide of processed mRNA, the mixture of total RNA (10 μg) and the Cy5.5-labeled oligonucleotide primer were denatured 5 min at 65 °C and subsequently incubated on ice for 2 min. Samples were reverse transcribed using 200 U of Hifair IV reverse transcriptase (Yeasen, China) in the presence of 5× first strand buffer, 5 mM dNTP Mix and 40 U of Recombinant Ribonuclease Inhibitor (Takara, Japan), as per the manufacturer’s instructions. Thermal cycling conditions were as follows: 25 °C for 5 min, 55 °C for 15 min and 85 °C for 5 min. The cDNA products were analyzed on an 8% polyacrylamide sequencing gel (8 M urea/TBE). Sequencing ladders were generated using USB Thermo Sequenase Cycle Sequencing Kit (Thermo Fisher Scientific, USA) with the same Cy5.5-labeled oligonucleotide primer, and the recombinant plasmids digested by BglII is as template.