GSM6171760: Group 3 innate lymphoid cells-KO rep3; Mus; RNA-Seq
Total RNA was extracted from the tissues using RNeasy Mini kit (QIAGEN, 74104) according to the manual instruction. Limited RNA（more than 200pg, high-quality）was amplified. Limited RNA（more than 200pg, high-quality）was amplified with oligo-dT and dNTPs,  incubated at 72℃ and immediately put back on ice, then reverse transcribed to cDNA based on   polyA tail. The template was switched to the 5' end of the RNA and the full-length cDNA was  generated 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 previous   steps was fragmented into small pieces with fragment buffer by PCR, and the product was purified   and selected by the Agencourt AMPure XP-Medium kit (Thermo Fisher Scientific, USA). cDNA   was quantified by Agilent Technologies 2100 bioanalyzer. The double stranded PCR product   undergo QC step was heat denatured and circularized by the splint oligo sequence. The single   strand circle DNA (ssCir DNA) was formatted as the final library. The final library was quantitated   in two ways in order to ensure the high quality of the sequencing data: Determined the average   molecule length used the Agilent 2100 bioanalyzer instrument, quantified library used real-time   quantitative 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) .