Pe150 sequencing mode

Total RNA was extracted from the uterine tissues of 12 ewes. Then, measurements of the RNA purity, concentration and integrity were conducted by 1% agarose electrophoresis, a Kaiao K5500 spectrophotometer (Beijing Kaiao Technology Development Co., Ltd., Beijing, China) and RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 System (Agilent Technologies, Santa Clara, CA, USA), respectively.
Library construction was performed using approximately 3 µg total RNA from each sample. Ribosomal RNAs (rRNAs) were removed from the total RNA using a Ribo-Zero™ Gold Kit (Epicentre, Madison, WI, USA). RNA libraries of 12 samples were generated using a NEB Next Ultra Directional RNA LibraryPrep Kit for Illumina (NEB, Ipswich, MA, USA), following the manufacturer’s instructions. CircRNAs were randomly fragmented and reverse transcribed into cDNA with random primers. Second-strand cDNA was synthesized using DNA polymerase I, RNase H, dNTPs, and buffer, and the cDNA fragment was purified by QiaQuick PCR, repaired at the end, tagged was a poly(A), and ligated into Illumina sequencing adapters. The samples were amplified by PCR and sequenced in the PE150 sequencing mode (Illumina, San Diego, CA, USA) using the Illumina X-Ten platform. Raw data of the RNA-Seq have been deposited in the SRA public database (Accession number: SRP173986).

The gel beads containing barcode information combine with a mixture of cells and enzymes,then enter the reservoir to be separated by oil to form GEMs (GelBeads-In-Emulsions). After that, the gel beads dissolved and released the capture sequence containing the Barcode sequence, reverse transcribed the cDNA fragment, and labeled the sample. Break up the gel beads and oil droplets then use cDNA as a template for PCR amplification. All the products of GEMs are mixed to construct a standard sequencing library. The cDNA was cut into 200 ∼ 300 bp fragments, then the second-generation sequencing library was constructed. Finally, the DNA library was amplified by PCR. The high-throughput sequencing of the library was carried out by using the PE150 sequencing mode of the Illumina sequencing platform. Quality control of sequencing was performed using cellranger.