GSM6533909: P7-Retinal-EC-Rnf20-Ctrl-RNAseq-Rep3; Mus musculus; RNA-Seq
PolyA RNA was isolated using Rneasy Universal Mini Kit from Qiagen BGI: BGISEQ-500 Ultra Low Input RNA Sample Library Construction Protocol: 1. Amplification Mix the PolyA RNA sample（more than 200 pg, good-quality）with oligo-dT and dNTPs. Incubate at 72℃ and immediately put it back on ice. Spin down the samples and reverse transcribed to cDNA based on polyA tail. Switch template at 5' end of the RNA and amplify the full-length cDNA by PCR. 2. Quality Control of Amplification Products Determine the average molecule length using the Agilent 2100 bioanalyzer instrument (Agilent High Sensitivity DNA Reagents). 3. Library Construction Follow Tagmentation-based library construction protocol. PCR products were purified and selected with the Agencourt AMPure XP-Medium kit. DNA was quantified by Agilent Technologies 2100 bioanalyzer. 4. Circularization The double stranded PCR products were heat denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were formatted as the final library. 5. Library quality control Library was qualified by the Agilent Technologies 2100 bioanalyzer. 6. Sequencing The library was amplified to make DNA nanoball (DNB) which had more than 300 copies of one molecular. The DNBs were loaded into the patterned nanoarray and single end 50 bases reads were generated in the way of sequenced by combinatorial Probe-Anchor Synthesis (cPAS) SMART-Seq Ⅱ