Poly a tail length assay kit

Poly(A) tail-length assay was performed using a Poly(A) tail-length assay kit (Thermo Scientific) according to the manufacturer’s instructions. In brief, total RNA was purified using TRIzol Reagent from HEK293T cells and treated with RNase-free DNase I to remove DNA. The G/I tailing mix was incubated with 2 μg RNA for 1 h at 37 °C. After incubation for 1 h, a tail stop solution was added. The RNA was then incubated with RT mix at 44 °C for 1 h and 92 °C for 10 min to synthesize cDNA. The cDNAs were amplified by PCR using PCR mix, DNA polymerase, and PCR primers. The specific oligonucleotides used in this study are listed in Supplementary Data 3c. The PCR products were loaded onto an agarose gel and stained with ethidium bromide.

A poly(A) tail assay was performed with a Poly(A) Tail-Length Assay Kit (764551KT, Thermo Fisher) according to the protocol. Briefly, poly(A) polymerase was used to add G and I to the 3′ ends of the RNA, and the newly tailed RNA was converted to cDNA by reverse transcription. Then, NOG-specific forward and reverse primers and universal reverse primers were used to generate a product consisting of NOG with a poly(A) tail. The PCR products from METTL3 knockdown DPSCs in differentiated/undifferentiated stages were separated on agarose gels [23 (link)]. The specific primers for NOG were F: 5′-TAACCTGCTATTTATATTCCAGTGCCCTTC-3′ and R: 5′-TGAACTCTATAGCTTCTTCGAGGTCCAA-3′.

RNA was extracted from S2 cells using either Ribosol (Amresco) or SIGMA RNA mini-prep per manufacturer’s protocol. Isolated RNA was DNase treated with Turbo DNase (Ambion) prior to cDNA synthesis. For cDNA synthesis 5x iScript Supermix (Bio-Rad) was used per manufacturer’s protocol. Quantitative PCR was performed with primers targeting Renilla luciferase or firefly luciferase, Supplemental Table 1. For 3′RACE: cDNA was synthesized using 3′RACE RT primer and 5x iScript Select (Bio-Rad) per manufacturer’s protocol. First round PCR was performed with Renilla-tail forward and 3′RACE External Amp primers. Second round PCR was performed with the overlap-forward primer for each 3′UTR being amplified (for example: GAPDH forward overlap) and 3′RACE amplification primer. The PCR products were resolved on an agarose gel. Prominent bands were excised and sequenced by Sanger sequencing. For qPCR of mature miRNAs we followed the protocol described previously^{65 (link)}. The primers used for this analysis are described in Table S3.
For analysis of p(A)-tail length we used the Poly(A) Tail-Length Assay Kit from Thermo-Fisher. The assay was performed per the manufacturer’s protocol. The primers used are described in Table S1: Hsp70a R, GAPDH R2 and HID/FLP F.

The analysis was performed based on a polyG/I extension method [47 (link)] using the Poly(A) Tail-Length Assay Kit (Thermo Fisher Scientific). In these experiments 5 µg/ml actinomycin-D (Sigma-Aldrich) was added to the medium of 16CAG and 98CAG Flp-In T-REx-293 cells to stop transcription. 200 ng of isolated RNA from selected time points were taken for poly(A) tail length analysis that was performed following manufacturer’s protocol. Specific primers used are listed in Table S4. For estimation of poly(A) tail lengths, a product obtained using gene-specific reverse primer was used as a reference. PCR products were analyzed on 2100 Bioanalyzer using DNA 1000 Kit (Agilent).

Total RNAs were isolated from embryos with indicated stages and genotypes using TRIzol reagent. The poly(A) tail length was tested with Poly(A) Tail-Length Assay Kit (ThermoFisher, 76455) following the manufacturer’s instructions. PCR products were analyzed on a 2% agarose gel. The detailed information of primers is described in Supplementary Table 3.