GSM6420006: RPE/choroid/slera complexes, control, 2; Mus musculus; ncRNA-Seq
Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA samples were first pretreated as following to remove some RNA modifications that interfere with small RNA-seq library construction: 3'-aminoacyl (charged) deacylation to 3'-OH for 3'-adaptor ligation, 3'-cP (2',3'-cyclic phosphate) removal to 3'-OH for 3'-adaptor ligation, 5'-OH (hydroxyl group) phosphorylation to 5'-P for 5'-adaptor ligation, m1A and m3C demethylation for efficient reverse transcription. Then, total RNA was sequentially ligated to 3' and 5' small RNA adapters. cDNA was synthesized and amplified using Illumina's proprietary RT primers and amplification primers. Subsequently, ~134-160 bp PCR amplified fragments were extracted and purified from the PAGE gel. And finally, the completed libraries were quantified by Agilent 2100 Bioanalyzer.