GSM7316555: mDC, 3mg, rpm, 2; Homo sapiens; RNA-Seq
RNA was extracted using TRIzol (Invitrogen, Thermo Fisher Scientific, Inc., Dreieich, Germany), followed by chloroform extraction (Sigma‒Aldrich, Schnelldorf, Germany) using the manufacturer's protocol. Afterward, purification using the RNeasy mini kit (Qiagen, Hilden, Germany) was performed according to the manufacturer's protocol. The obtained RNA concentration and purity (the ratio of absorbance at 260 nm and 280 nm) were quantified using a Nanodrop (Thermo Fisher Scientific, Inc., Dreieich, Germany) and confirmed by a Qi RNA kit using a Qubit 4 fluorometer (Thermo Fisher Scientific, Inc., Dreieich, Germany) prior to performing RNA sequencing. Purified RNA samples were prepared with an NEB Ultra II RNA kit (New England Biolabs, Ipswich, MA, USA) according to the protocol instructions using the NEBNext Poly(A) mRNA Magnetic Isolation module (New England Biolabs, Ipswich, MA, USA) and uniquely dual indexed. The resulting library concentration, size distribution, and quality were assessed on a Qubit 4 fluorometer (Thermo Fisher Scientific, Inc., Dreieich, Germany) with a dsDNA high sensitivity kit (Invitrogen, Carlsbad, CA, USA) and on a 4200 TapeStation using a High Sensitivity D5000 kit (Agilent, Santa Clara, CA, USA). Based on these results, libraries were normalized according to their molarity and pooled and then quantified with a library quantification kit for Illumina platforms (Roche, Basel, Switzerland) on a StepOnePlus qPCR machine (Thermo Fisher Scientific, Inc., Dreieich, Germany). Finally, pooled libraries were loaded at 350 pM with 1% PhiX on S2 FlowCell and paired end sequenced (2 × 150 bp) on a NovaSeq 6000 next generation sequencer (Illumina, San Diego, CA, USA). RNA-Seq was performed in triplicate.