title: GSM3819866: NBM-11-CD1c; Homo sapiens; RNA-Seq
library_construction_protocol: < 100 cells were directly sorted into PCR tubes containing 9.5 µL of PBS. RNA-seq libraries were prepared using the SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio USA, Inc.). Briefly, the cells were lysed and first-strand cDNA was synthesized utilizing template switching oligos. Amplified double stranded cDNA was created by PCR using blocked PCR primers. The resulting cDNA was purified using AmpureXP beads (Beckman Coulter, Inc). Purified cDNA was quantitated by Qubit hs DNA (Thermo Fisher Scientific, Inc.) and 1ng of material was used for subsequent library generation utilizing Nextera XT reagents (Illumina, Inc). Tagmentation was performed to shear the full length cDNA to the appropriate size and incorporate partial adapter sequences. 12 cycles of PCR using Nextera XT primers complete the library and incorporate unique sample barcodes. The final libraries were purified using AmpureXP beads, and validated for appropriate size on a 4200 TapeStation D1000 Screentape (Agilent Technologies, Inc.). The DNA libraries were quantitated using KAPA Biosystems qPCR kit, and were pooled together in an equimolar fashion.  Each pool was denatured and diluted to 2.4pM with 1% PhiX control library added.  The resulting pool was then loaded into the NextSeq 150 cycle high output reagent cartridge, and sequenced on a NextSeq500 following the manufacturer's recommended protocol (Illumina, Inc.).