title: GSM4774308: PEM-78_2 RNA-seq; Homo sapiens; RNA-Seq
library_construction_protocol: Cells of about 10^5-10^6 in total are disrupted in 350 microliter RLT buffer with the addition of  beta-mecaptoenthanol and then homogenized as described in the kit (RNeasy mini kit, Qiagen Cat # 74104). 70% Ethanol is added to the lysate with the volume ratio 1:1 which is then followed by column purification steps. The residual amount of DNA remaining is removed using on-column DNase treatment and RNA is eluted in 30 µl of nuclease-free water. SmartSeqHT: According to the manufacturer's protocol, amplified cDNA was generated by using the SMARTer HT RNA kit (Clontech/Takara) followed by Nextera® XT DNA Library Preparation kit (Illumina) for sample barcoding and fragmentation. Briefly, 10 ng of RNA was subjected to first‐strand synthesis of RNA molecules was performed using the poly‐dT‐based 3'‐SMART CDS primer II A (Clontech) followed by extension and template switching by the reverse transcriptase. The second strand synthesis and amplification polymerase chain reaction (PCR) was run for 18 cycles, and the amplified cDNA was purified with a 1× Agencourt AMPure XP bead cleanup (Beckman Coulter,). The Nextera XT DNA Library Preparation kit (Illumina) was used for sample barcoding and fragmentation according to the manufacturer's protocol. One nanogram of amplified cDNA was used for the enzymatic tagmentation, followed by 12 cycles of amplification and unique dual‐index barcoding of individual libraries. PCR product was purified with a 1.8× Agencourt AMPure XP bead cleanup (Beckman Coulter).