GSM6421726: Neutrophils_Trt6hr-3 [Sample6]; Mus musculus; RNA-Seq
Each of the cell type was either untreated or treated with 500 ng/ml of rmIL-36γ (R&D Technologies) for 6h or 24h and RNA from extracted using RNeasy isolation kit (Qiagen) according to manufacturer's guidelines. Depending on the RNA amount available, 0.7- 5.0 ng of total RNA was used as the starting input to generate the full-length cDNA by using the SMART-Seq v4 Ultra-low Input RNA Kit for Sequencing (Takara Catalog No.634890). RNA was primed by the 3′ SMART-Seq CDS Primer II A with the SMART-Seq v4 Oligonucleotide for template switching at the 5′ end of the transcript during the first-strand cDNA synthesis. Preparation of SMARTer cDNA was sequentially amplified by following the LD-PCR procedure, which was carried out via the thermal cyclic program according to the input amount of total RNA: 11 PCR cycles for 1.0-5.0ng RNA samples and 15 PCR cycles for 0.7-1.0 ng RNA samples. The LD-PCR amplified cDNA was purified and further validated in the Agilent High Sensitivity D5000 ScreenTape (Agilent, Catalog No.5067-5592) on the Agilent 4200 TapeStation. Then, 300pg cDNA products were used for library preparation in each reaction by using the Illumina Nextera XT kit and following the manual instruction (Illumina, Catalog No.FC-131-1096). The products of low-input cDNA libraries were then barcoded after by 12 cycles of thermal program with specific index adapters for Illumina sequencing (Nextera XT Index Set D, Illumina, Catalog No. FC-131-2004). The resulting libraries were cleaned up using 0.8x AMPure XP beads (Beckman Coulter, Catalog No. A63881) and eluted in 25 µl of resuspension buffer. After library QC and quantitation, libraries were sequenced to a minimum depth of 45 million paired-end/Dual-indexed 2x150bp reads on an Illumina HiSeq4000).