Sequel 2 system

To generate the SMRTbell libraries, we combined equal amounts of total RNA from the biological replicates and generated an RNA pool for SMRT sequencing. From this pool, oligo(dT) was used to enrich for mRNA containing a poly-A tail, and the mRNA was reverse transcribed into cDNA using a SMARTer PCR cDNA Synthesis Kit. We used PCR to amplify the cDNAs. The fragments were then screened for large-scale PCR to obtain sufficient cDNA. The resulting full-length cDNA was subjected to injury repair, end-repair, ligated to SMRT dumbbell-type linkers, and used to construct a full-length transcriptome library. We removed the unligated linker sequences at both ends of the cDNA, added primers, and used DNA polymerase to form a complete SMRTbell library.
The library was sequenced using the PacBio Sequel II System and SMRT. The raw Iso-Seq data were processed with SMRTlink v6.0 software to obtain subread sequences. CCSs were obtained following correction between subreads. Full-length sequences containing a 5′ primer, a 3′ primer, and a poly-A tail were clustered using the Iterative Isomer Clustering (ICE) algorithm. Finally, the resulting consensus sequences were calibrated using the clean reads to obtain high-quality sequences for subsequent analysis.

A single human oocyte or embryo (details in Fig. 1b) was washed with 1× phosphate-buffered saline (PBS, Invitrogen, AM9625) containing 0.1% bovine serum albumin (BSA, Sigma-Aldrich, A1933) three times, and transferred into a 0.2-ml thin-walled PCR tube containing 2.5 µl of cell lysis buffer (0.2% Triton X-100 (Sigma-Aldrich, T9284) containing 2 U/µl of RNase inhibitor (TaKaRa, 2313A)) using a micro capillary pipette in the lowest possible volume (around 0.5 µl) to a final volume of around 3 µl. Then samples were incubated at 85 °C for 5 minutes for lysis and denaturation of the RNA, then put on ice immediately. The single-oocyte/embryo PAIso-seq library construction was carried out following our recently published detailed protocol^{40 (link)}. The libraries were sequenced on a PacBio Sequel or Sequel II System under HiFi mode according to the standard PacBio Iso-Seq procedures at Annoroad (a sequencing service provider in China, http://www.annoroad.com/).

Four samples (pre-treatment and relapse samples from patients 3 and 4) were sequenced on the PacBio Sequel II system using 8M SMRTCells for 30 hours. The remaining samples were sequenced on the PacBio Revio system using Revio SMRT Cells for 24 hours. Raw HiFi reads were segmented into representative cDNAs with SMRT Link v12.0.0.177059 using the 10X Chromium single cell 3’ cDNA primers barcode set and MAS-Seq Adapter v1 (MAS16) barcode set. We used PacBio’s Iso-Seq single-cell workflow to trim, tag, and align reads using the default parameters of the tools distributed via Bioconda (https://github.com/PacificBiosciences/pbbioconda). Briefly, Lima v2.7.1 removed primers from the segmented reads. Isoseq v4.0.0 added tags for unique molecular identifiers (UMIs) and barcodes, followed by trimming of the PolyA and primer sequences. Isoseq was then used to correct cell barcodes and deduplicate reads. Finally, pbmm2 v1.10.0 aligned reads to the reference genome using the GRCh38 reference sequence. Pigeon v1.2.0 generated Seurat-compatible files for downstream transcriptomic analysis.