GSM5506057: GNS166 shL2A exp3; Homo sapiens; RNA-Seq
mRNA samples were extracted by TRIzol technique. For library construction, RNA was extracted from a sample first. After DNase I treatment and performing quality control (QC), 200 ng of high-quality total RNA was proceed to library construction. Magnetic beads with Oligo (dT) are used to isolate mRNA. The mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer after adding dNTPs, RNase H and DNA polymerase I. Short fragments are purified and resolved with EB buffer for end repair and single nucleotide A (adenine) addition. After that, the short fragments are connected with sequencing adapters. The double-stranded cDNA library is completed through size selection and PCR enrichment. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used in quantification and qualification of the sample library. Lastly, the qualified RNA-seq libraries are sequenced using Illumina NovaSeq6000 in CD Genomics (Shirley, NY) after pooling according to its effective concentration and expected data volume.