Nextera xt dna sample preparation index kit

Single-cell loading, capture, and library preparations were performed using the Fluidigm C1 system as described previously [25 (link)]. 5000–8000 cells were loaded onto a medium-sized (10–17 μm) C1 Single-Cell Auto Prep IFC (Fluidigm) and the cell-loading script was used according to the manufacturer’s instructions. The capture efficiency was inspected using EVOS FL Auto Cell Imaging system (Life Technologies) to perform an automated area scanning of the 96 capture sites on the IFC. Empty capture sites or sites having more than one cell captured were first noted, and those samples were later excluded from further library processing for RNA-seq. Immediately after capture and imaging, reverse transcription and cDNA amplification were performed in the C1 system using the SMARTer PCR cDNA Synthesis Kit (Clontech) and the Advantage 2 PCR Kit (Clontech) according to the instructions in the Fluidigm user manual. Full-length, single-cell cDNA libraries were harvested the next day from the C1 chip and diluted to a range of 0.1–0.3 ng/μL. Diluted single-cell cDNA libraries were fragmented and amplified using the Nextera XT DNA Sample Preparation Kit and the Nextera XT DNA Sample Preparation Index Kit (Illumina). Libraries were multiplexed at 24 or 48 libraries per lane, and single-end reads of 67 bp were sequenced on an Illumina HiSeq 2500 system.