GSM2409500: O_K_1_sRNA; Carassius gibelio; miRNA-Seq
RNA preparation, library construction and high-throughput sequencing were performed by LC Sciences (Houston, TX, USA). The experimental procedure is introduced as follows: total RNAs were extracted using Trizol reagent (Invitrogen, CA, USA) following the instruction of the product manual. The total RNA quantity and purity were analyzed by using Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number higher than 7.0. The total RNA with lowest quality was not used for further study from the pre-receptive endometrium samples and receptive endometrium samples, respectively. For sRNA-seq experiment, approximately 1 μ g of total RNA was subjected to construct sRNA library according to the protocol of TruSeq™ Small RNA Sample Prep Kits (Illumina, San Diego, USA). Then, single-end sequencing (50 bp) was performed on the Illumina Hiseq2500 platform following the vendor’s recommended protocol. For RNA-seq experiment, approximately 10 μ g of total RNA was used to enrichment of poly (A)-tailed mRNAs with poly-Toligo attached magnetic beads (Invitrogen, MA, USA). Following purification, the mRNAs were fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments were reverse-transcribed to produce the final cDNA library according to the protocol of the mRNA-seq sample preparation kit (Illumina, San Diego, USA). Each PE and RE library was constructed by pooling 9 homogenized total RNAs from the endometrium samples. Then, paired-end sequencing of the PE and RE libraries was performed on the Illumina Hiseq2500 (LC Sciences, USA) following the vendor’s recommended protocol. The length of the reads was 100 bp, and the average insert size for the paired-end libraries was 179 bp (the length of the adapter was 121 bp). Pre-treatment of the sRNA-seq. First, the sequencing adapters were removed and the short reads containing ambiguous base “N” were discarded. Then, in order to allow cross-library comparison, read count normalization was performed. Specifically, the normalized read count (in RPM, reads per million) of a short sequence from a sequencing library was calculated by dividing the raw count of this sequence by the total counts of the library, and then multiplied by 10^6.
GEO Accession
GSM2409500