GSM8013684: peripheral blood, P1; Homo sapiens; RNA-Seq
Peripheral blood was obtained from healthy volunteers. Mononuclear cells were obtained using Lymphoprep (Serumwerk, Berunburg, Germany), according to the manufacturer's protocols. Lineage-negative (CD3, CD14, CD16, CD19, CD20 and CD56) and CD45-, CD127-, CRTH2- and CD161-positive cells were sorted using a MoFlo XDP flow cytometer (Beckman Coulter, Brea, CA, USA) as ILC2s. We obtained cDNA from poly(A) RNA of each sample using a SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio Inc., Shiga, Japan), following the manufacturer's instructions. The pre-amplified cDNA was obtained after reverse transcription (90 min at 42 °C, 10 min at 70 °C) and pre-amplification (24 cycles (N1–N4) or 18 cycles (P1–P3): 1 min at 95 °C, 10 s at 98 °C, 30 s at 65 °C, and 3 min at 68 °C). After the purification of the amplified cDNA using Agencourt AMPure XP beads (A63881, Beckman Coulter), cDNA sequences were analyzed using Illumina HiSeq with an entrusted analysis service (50 bp single-read, Medical & Biological Laboratories Co., Ltd. Japan).