GSM4547649: M13_CIITA_DC_3_RNAseq; Homo sapiens; ncRNA-Seq
RNA was extracted using miRNeasy Mini Kit (Qiagen). The following QC and library construction steps wer performed at Novogene using the lncRNA-seq pipeline. Nanodrop was used for a preliminary quantitation of the RNA concentration. Then agarose gel electrophoresis was performed to test the RNA degradation and potential contamination and Agilent 2100 was used to test the RNA integrity and concentration quantification. rRNA was removed using the Ribo-Zero kit. First, the mRNA was fragmented randomly by adding fragmentation buffer, then the cDNA was synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina), dNTPs (dUTP, dATP, dGTP, and dCTP), RNase H and DNA polymerase I were added to initiate the second-strand synthesis, followed by purification by AMPure XP beads, terminal repair, polyadenylation, sequencing adapter ligation, size selection, and degradation of second-strand U-contained cDNA by the USER enzyme. The strand-specific cDNA library was generated after the final PCR enrichment. The library quality control consisted of three steps: (1) Qubit 2.0: tests the library concentration preliminarily, (2) Agilent 2100: tests the insert size, (3) Q-PCR: quantifies the library effective concentration precisely. The qualified libraries were then fed into Illumina sequencers (Illumina Novaseq 6000) after pooling according to their effective concentration and expected data volume with paired end 150 bp sequencing strategy.
GEO Accession
GSM4547649