Acgt101 mir

Raw reads of the miRNA were examined using the software ACGT101-miR (v4.2, LC Sciences, Houston, TX, USA) to remove the sequences with splice contamination, unidentifiability, and a base ratio greater than 10% in the raw data. Among the remaining sequences, those with 18–32 nucleotides were retained. Thereafter, a variety of RNA databases, including the mRNA database, Repbase database https://www.girinst.org/ (accessed on 26 May 2022), and RFam database (v14.7) https://rfam.xfam.org (accessed on 26 May 2022), were used to compare and filter the remaining sequences (apart from miRNA). The final valid data were used for a subsequent analysis of the miRNA. The effective sequence was mapped to the reference full-length transcriptome of silver carp, and miRBase (v22.0) https://mirbase.org/ (accessed on 26 May 2022), was used to obtain known miRNAs. For secondary structure prediction, the mfold program was utilized to obtain novel miRNAs. Finally, the Ensembl and miRBase databases were used to annotate the sequences of known and novel predicted miRNAs.

Raw sequencing reads were analyzed by the ACGT101-miR program (LC Sciences, Houston, TX, USA). After removing the 3′ adapters and junk sequences, the remaining sequences with a length of 18–25 nt were aligned to mRNA (http://rapdb.dna.affrc.go.jp/download/irgsp1.html, accessed on 3 September 2022), Rfam (http://rfam.janelia.org, accessed on 3 September 2022), and repeat (http://www.girinst.org/repbase, accessed on 3 September 2022) databases to remove the matched sequences, respectively. The remaining valid reads were blasted against miRbase (http://www.mirbase.org/, accessed on 3 September 2022) [62 (link)] to identify the known miRNAs. The remaining unmapped sequences were compared with the rice genome (http://rapdb.dna.affrc.go.jp/download/irgsp1.html, accessed on 3 September 2022), and mapped sequences that fulfilled the criteria for the annotation of plant miRNAs were identified as novel miRNAs [63 (link)]. The p value of the Student’s t-test was used to analyze the DEMs based on normalized deep-sequencing counts; the p-value ≤ 0.05 was set as the significance threshold of DEMs in this test.

Total RNA was extracted from every sample using Trizol reagent (Invitrogen, CA, USA) following the manufacturer’s procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 (Agilent, CA, USA) and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number> 7.0. Approximately 1 ug of total RNA were used to prepare small RNA library according to protocol of TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, USA). The total RNA with lowest quality was not used for further study from the pre-receptive endometrium samples and receptive endometrium samples, respectively. P library was constructed by mixing the nine samples into three samples with the same concentration, performing the single-end sequencing (36 bp) on an Illumina Hiseq2500 (Illumina, San Diego, USA) thrice at the LC-BIO (Hangzhou, China) following the vendor’s recommended protocol, and R library was constructed in the same way.
Data processing followed the procedures described in a previous study [69 (link)] by LC Sciences Service. Briefly, the raw reads were subjected to the Illumina pipeline filter (Solexa 0.3), and then the dataset was further processed with an in-house program (ACGT101-miR, LC Sciences, Houston, Texas, USA) to remove adapter dimers, junk, low complexity, common RNA families (rRNA, tRNA, snRNA and snoRNA) and repeats.