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2ug total RNA was used for cDNA library preparation by using a modified protocol based on the Illumina Truseq RNA Sample Preparation Kit V2. After poly-A selection, fragmentation, and priming, reverse transcription was carried out for 1^st strand cDNA synthesis in the presence of RNaseOut (Invitrogen) and actinomycin-D (MP Biomedicals). The synthesized cDNA was further purified by using AMPure RNAClean beads (Beckman Coulter). A modified method by incorporation of dUTP instead of dTTP was prepared and used for the second strand synthesis. After AMPure XP bead purification (Beckman Coulter), following the standard protocol recommended by the Illumina Truseq RNA kit, end repairing, A-tailing, and ligation of index adaptors were sequentially performed for generation of cDNA libraries. After size selection of libraries using Pippin Prep (SAGE Biosciences), the dUTP-containing strands were destroyed by digestion with USER enzymes (New England Biolabs) followed by PCR enrichment. Final cDNA libraries were analyzed in Agilent Bioanalyzer and quantified by Quant-iT Pico-Green assays (Life Technologies) before sequencing using the HiSeq platform (Illumina).

Single GV oocyte was washed by using PBS with 1% BSA three times. Single GV oocyte was lysed in buffer containing 0.5 μl of RNase inhibitor to 10.5 μl of 0.2% (vol/vol) Triton X-100 at 80 °C for 5 min. The mixture was then used for end extension with dNTP and Klenow fragment (exo⁻, NEB) on a dU-containing DNA template (Supplementary Table 1) at 37 °C for 1 h. Fifteen single GV oocytes were barcoded with different dU-containing DNA templates (Supplementary Table 1). dUs were digested by USER enzymes (NEB) at 37 °C for 30 min. End-extended RNAs (>200 nt) was cleaned and concentrated with RNA Clean & Concentrator-5 Kit (Zymo Research). Fifteen cleaned end-extended RNAs were combined and proceeded with regular PAIso−seq library construction.

The Amgen cDNA libraries were prepared from total RNA isolated using Mirvana miRNA isolation kits (Ambion, Grand Island, NY) with on-colum DNase treatment as described previously60 (link). Total RNA quality and concentration was determined using Bioanalzyer (Agilent, Santa Clara, CA) and NanoDrop (ThermoScientific, Wilmington, DE) instruments. cDNA was prepared using a modified protocol based on the Illumina TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA) and published methods for strand-specific RNA-Seq61 (link)62 (link). After size-selection of libraries (Pippen Prep; SAGE Biosciences, Beverly, MA), dUTP-containing cDNA strands were eliminated by digestion with USER enzymes (New England BioLabs, Ipswich, MA) followed by PCR enrichment for introduction of strand specificity. The enriched cDNA libraries were analyzed in Agilent Bioanalzyer and quantified by Quant-iTTM Pico-Green assays (Life Technologies). The samples were sequenced on the Illumina HiSeq platform (Expression Analysis Inc, Morrisville, NC) using 75 bp paired-end sequencing to a depth of 35–55 million reads.

A sequencing library was prepared with 3 µg of RNA from each sample using a NEBNext Ultra RNA Library Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. The RNA was purified and broken into small random fragments using poly-T oligo attached magnetic beads (Life Technologies, Carlsbad, CA, USA). Double-stranded DNA was synthesized using a TruSeq™ Stranded mRNA Prep Kit (Illumina). The DNA fragments in the library with a length of 150–200 bp were screened and purified using an Ampure XP system (Beckman Coulter, Beverly, MA, USA). The purified double-stranded cDNA (size selection and connection) was incubated with 3 ml of USER enzymes (NEB, Ipswich, MA, USA) at 37 °C for 15 min and then cultured at 95 °C for 5 min. Polymerase chain reaction (PCR) was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers, and index (X) Primer, and the products were then purified using the Ampure XP system. The composite samples were paired and sequenced using a HiSeq™ 25,000 for 2 × 100 bp according to the manufacturer’s instructions. The PE reading of each lane was about 150 m (n = 3).

RNA was extracted using the RNEasy Mini kit with gDNA eliminators columns (QIAGEN). An RNA-Seq experiment was performed with 500 ng total RNA as starting material (n = 2 for each cell type), to obtain double-stranded cDNA as previously described^{71 (link)}. After purification with the MinElute Reaction Cleanup Kit (28206, QIAGEN), 3 ng of ds-cDNA were processed for library construction using the KAPA Hyper Prep Kit (KK8504, KAPA Biosystems, Roche) according to the standard protocol except that a 15 min USER enzyme (M5505L, NEB, Ipswich, MA, USA) incubation step was added before library amplification. The prepared libraries were quantified with the KAPA Library Quantification Kit (KK4844, KAPA Biosystems, Roche), and then sequenced in a paired-ended manner using a NextSeq 500 (Illumina) according to standard Illumina protocols. Approximately 15–25 × 10⁶ reads were sequenced for each sample. Sequencing reads were aligned to human genome assembly hg19 (NCBI version 37) using STAR 2.5.0 with default options. Genes with the mean of DESeq2-normalized counts (“baseMean”) > 10 were considered to be expressed^{72 (link)}. Differential gene expression was defined as adjusted P value (padj) < 0.05, and fold change > 2. Functional gene ontology annotation of genes was performed with DAVID^{73 (link)}.

According to the reported protocol, six RNA libraries for input data were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, Ipswich, MA, USA). Poly-T oligo-attached to magnetic beads were used to extract mRNA from the prepared RNA library. In the NEBNext First Strand Synthesis Reaction Buffer, fragmentation was performed five times with divalent cations at a high temperature. M-MuLV Reverse Transcriptase and random hexamer primers were used to make first-strand cDNA (RNase H-). Following that, DNA polymerase I and RNase H were used to synthesize second-strand cDNA. The library fragments were purified using the AMPure XP technology to select cDNA fragments ranging from 150 to 200 bp (Beckman Coulter, Beverly, USA). Before PCR, 3 μl of USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 minutes and then 5 minutes at 95°C. Phusion High-Fidelity DNA Polymerase, universal PCR primers, and index (X) primer were used in the PCR. According to the manufacturer's instructions, the prepared library quality was assessed using the Agilent Bioanalyzer 2100 system, and PCR products were purified (AMPure XP system). The TruSeq PE Cluster Kit v3-cBot-HS (Illumina) was used to cluster the index-coded samples on a cBot Cluster Generation System.

Three micrograms RNA was used for the RNA sample preparations. Sequencing libraries were produced using NEBNext^® Ultra™ Directional RNA Library Prep Kit for Illumina^® (NEB, Ipswich, MA, USA), and index codes were added to sequences. NEBNext First Strand Synthesis Reaction Buffer (5×) was used with divalent cations at a high temperature to obtain fragmentation. The first-strand cDNA was synthesized by M-MuLV Reverse Transcriptase (RNase H, NEB, Ipswich, MA, USA) and random hexamer primer (NEB, Ipswich, MA, USA). The second-trand cDNA was synthesized with RNase H and DNA Polymerase I. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. The AMPure XP system (Beckman Coulter, Beverly, MA, USA) was used to screen cDNA fragments of 150~200 bp in length to purify the library fragments. Then 3 µL USER Enzyme (NEB, USA) was used with cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then, the reaction was carried out with Phusion High-Fidelity DNA polymerase kit (NEB, Ipswich, MA, USA), Universal PCR primers and Index (X) Primer. Finally, the Agilent Bioanalyzer 2100 system (G2939B, Agilent Technologies, Palo Alto, CA, USA) was used to assess products and library quality.