GSM4432261: c50_1; Homo sapiens; RNA-Seq
For the RNA extraction, cells collected in Cellotion were pelleted and lysed and stored at −80°C until use. RNA extraction was performed with RNeasy Micro Kit (QIAGEN, 74004) or NucleoSpin RNA XS (MACHEREY-NAGEL, U0902A) according to the manufacturer's instruction. When using NucleoSpin RNA XS, we added an additional centrifugation just before the elution step. The concentration of RNA was measured with Qubit RNA HS Assay Kit (Invitrogen, Q32855). 1 ng of total RNA from each sample was used for the reverse transcription and the cDNA amplification was performed as described previously. When the RNA concentration was too low to be measured (in the present study, RNA extraction was performed with 1,606 to 6,516 cells), we centrifuged the RNA solution to reduced its volume and used the whole concentrated RNA solution for the reverse transcription and the cDNA amplification. For RNA-seq analysis, sequence libraries were constructed with amplified cDNAs as described previously (Nakamura et al., 2015; Ishikura et al., 2016) with some modifications. When purifying cDNA libraries, we used Axyprep MAG PCR Clean-up Kit (Axygen, MAG-PCR-CL-250), instead of Agencourt AMPure XP (Beckman Coulter, A63881). Sequences were performed on Illumina NextSeq 500/550 platform with NextSeq 500/550 High output kit v2.5 (75 cycles) (Illumina).