Dynabeads mrna direct kit

As previously described^{42 (link)}, mRNA was extracted from 30 embryos in each group using a Dynabeads mRNA Direct Kit (61012; Thermo Fisher Scientific, Waltham, MA, USA), and cDNA was obtained using a First Strand Synthesis Kit (cat# 6210; LeGene, San Diego, CA, USA) following the manufacturer’s instructions. qRT-PCR was performed using a WizPure qPCR Master mix (W1731-8; Wizbio Solutions, Seongnam, South Korea) according to the manufacturer’s instructions, using a QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). Amplification was performed as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of amplification at 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 15 s, and a final extension at 95 °C for 15 s. Relative gene expression was calculated using the ∆∆CT method. The primers used to amplify each gene are listed in Table 1.Table 1

Primer sequences used in RT-qPCR.

Gene	Gene accession no	Primer sequence (5′–3′)	Annealing temp (°C)	Length (bp)
GRP78	J03214.1	F: ACC AAT GAC CAA AAT CGC CT R: GTG ACT TTC CAG CCA CTC AA	60	246
ATF4	NM_001123078.1	F: TGA GCC CTG ACT CCT ATC TG R: TCC AGC TCT TTA CAT TCG CC	60	277
CHOP	NM_001144845.1	F: AAG ACC CAG GAA ACG GAA AC R: TCC AGG AAA GGT CAG CAG TA	60	261
GAPDH	AK234838	F: AAGTTCCACGGCACAGTCAAG R: CACCAGCATCACCCCATTT	60	112

The Dynabeads® mRNA Direct kit (Thermo Fischer Inc.) was used to purify mRNA from 5 μg of total RNA according to the manufacturer’s instructions. We used the Ion Xpress Plus Fragment Library Kit (Thermo Fischer Inc.) to prepare a cDNA library according to the manufacturer’s instructions. Samples were sequenced using Ion Proton semiconductor sequencers (Thermo Fischer Inc.) according to the manufacturer’s instructions. We aligned raw reads to the mouse reference genome (Ensembl, mm9) using the spliced read aligner STAR^{45 (link)}, then took any unmapped reads to Bowtie2 in the local mode^{46 (link)}. Raw read counts were calculated by featureCounts^{47 (link)} in the Subread package. The detection of DEG and calculation of rpkm (reads per kilobase of exon per million mapped sequence reads) were performed using the edgeR package^{48 (link)} with Ensembl genes, filtered against protein-coding genes. In order to visualize gene expression, the rpkm of genes was log2 transformed with primary counts of 0.25, mean centered between Nrdc−/− and Nrdc−/−^WT iMEF, and plotted with the gplots package.

mRNA was extracted from 35 COCs after 24 h of culture in IVM medium using a Dynabeads mRNA Direct Kit (61,012; Thermo Fisher Scientific, Waltham, MA, United States), and cDNA was synthesized using the First Strand Synthesis Kit (cat# 6210; LeGene, San Diego, CA, United States) in accordance with the manufacturer’s instructions. The primer sequences for amplification of cDNA were the same as those used in previous studies (Han et al., 2016 (link); Park et al., 2018 (link)). qRT-PCR was performed using a WizPure qPCR Master (W1731-8; Wizbio Solutions, Seongnam, South Korea) according to the manufacturer’s instructions, on a QuantStudio™ six Flex Real-Time PCR System (Applied Biosystems, Waltham, MA, United States). The PCR conditions were as follows: initial denaturation at 95°C for 10°min, followed by 40 cycles of amplification at 95°C for 15°s, 60°C for 20°s, and 72°C for 15°s, and a final extension at 95°C for 15°s. Relative gene expression was calculated using the ∆∆CT method. The primers used are listed in Table 1.

Unsynchronized HeLa cells with 90% confluency in 15 cm plates were collected by cell lifters, pelleted by centrifuge for 5 minutes at 400g, and washed once with cold PBS. The cell pellets were lysed with 3 volumes of lysis buffer (150 mM KCl, 10 mM HEPES [pH 7.6], 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:50 protease inhibitor cocktail, 1 U/μL SUPERase•In RNase Inhibitor) on ice for 20 minutes. The lysates were centrifuged at 16,000g for 15 minutes; 50 μL cell lysate was saved as input and mixed with 1 mL TRIzol for RNA extraction; and 20 μg of YTHDF2 antibody was added to the rest of the lysate and incubated by rotating for 2 hours at 4°C. Afterward, 40 μL of Pierce Protein A/G Magnetic Beads was washed twice with lysis buffer and added to the lysate and was incubated by rotating for 1 hour at 4°C. After separation using a magnetic rack, beads were washed with ice-code lysis 6 times. Beads were then mixed with 1 mL TRIzol and recovered for RNA, which was saved as immunoprecipitation. RNA extracted from input was further subjected to mRNA purification using the Dynabeads mRNA DIRECT kit (Thermo Scientific). Input mRNA of 100 ng and immunoprecipitation RNA samples with 2 biological replicates were used for RNA-seq library preparation.

Cells were cultured in six well plate format in triplicates and lysed in 500 µL Dynabeads mRNA lysis buffer when about 70% confluent. Invitrogen Dynabeads mRNA Direct Kit (Cat # 610-12; Thermofisher Scientific, Oxford, UK) was used for mRNA extractions from the cells. A total of 50 ng mRNA from each cell line was used to prepare the cDNA with a mixture of oligo-dT and random hexamers using the qPCRBIO cDNA Synthesis Kit (Cat # PB30.11-10, PCR Biosystems, London, UK) in triplicates according to the manufacturer’s instructions. For total RNA extraction, Qiagen RNeasy Kit (Cat # 74104) was used according to the manufacturer’s protocol. The reverse transcription reaction was carried out at 42 °C for 30 min, heat inactivated at 85 °C for 5 min and cooled to 4 °C for 5 min. cDNA was diluted 1:20 with RNase/DNase-free water and stored at −80 °C until used.

For MeRIP-seq, total RNA was isolated using TRIzol reagent. The obtained mRNA was further purified using the Dynabeads mRNA DIRECT Kit (Thermo Fisher) and fragmented by sonication. MeRIP-seq and library preparation were performed as per the reported protocol³³ (link) with some modifications. In brief, sonicated mRNA was mixed with m⁶A antibody (Synaptic Systems, 202003) in IP buffer and incubated under head-to-tail mixing at 4°C for 2 h. The mixture was supplemented with protein A magnetic beads (Thermo Fisher) and incubated under head-to-tail mixing at 4°C for another 2 h. The beads were then washed with IP buffer three times before elution with m⁶A elution buffer two times. The eluates were combined and purified by an RNA Clean and Concentrator (Zymo, Orange, CA). The purified mRNA fragments were used to construct libraries with the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA). Sequencing was carried out on the Illumina HiSeq 2000 system with pair-end 150-bp read length. Reads were aligned to human genome version 38 (GRCh38) with TopHat. The longest isoform was retained if a gene had more than one isoform. Differential m⁶A-modified peaks between IP and input samples were identified using exomePeak (p < 0.01).