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Small intestinal ILC3 subsets were sorted from siLP of Utxflox/floxJmjd3flox/flox mice for RNAseq. Limited RNA (more than 200pg, high-quality) was extracted and amplified with oligo-dT and dNTPs.incubated at 72'C and immediately put back on ice, then reverse transcribed to cDNA based onpolyA tail. The template was switched to the 5' end of the RNA and the full-length cDNA wasgenerated by PCR. Agilent 2100 bioanalyzer instrument (Thermo Fisher Scientific, MA. USA)was used to determine the average molecule length of PCR product. Purified cDNA from previoussteps was fragmented into small pieces with fragment buffer by PCR, and the product was purifiedand selected by the Agencourt AMPure XP-Medium kit (Thermo Fisher Scientific, USA). cDNAwas quantified by Agilent Technologies 2100 bioanalyzer. The double stranded PCR productundergo OC step was heat denatured and circularized by the splint oligo sequence. The singlestrand circle DNA (ssCir DNA) was formatted as the final library. The final library was quantitatedin two ways in order to ensure the high quality of the sequencing data: Determined the averagemolecule length used the Agilent 2100 bioanalyzer instrument, quantified library used real-timequantitative PCR (qPCR).The final library was amplified with phi29(Thermo Fisher Scientific.MA, USA) to make DNA nanoball (DNB) which had more than 300 copies of one molecular .DNBs were loaded into the patterned nanoarray and single end 50 bases reads were generated on BGISEQ500 platform (BGI-Shenzhen, China). |
RNA-Seq |
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size fractionation |
SINGLE
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