Smarter ultra low rna kit for the fluidigm c1 system

Isolated Venus⁺ osteoblasts (300 cells/μL) were loaded onto the C1 Single‐Cell mRNA Seq IFC (10‐ to 17‐μm cell diameter; Fluidigm, South San Francisco, CA, USA), and captured single cells were confirmed by phase‐contrast microscopy to exclude doublets and debris from further analysis. cDNAs were prepared in integrated fluidic circuits using the SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (Takara Bio, Shiga, Japan). A bulk control (about 100 to 200 cells) and a negative (no template) control were processed in parallel using the same reagents and methods. Sequencing libraries were constructed in 96‐well plates using the Nextera XT DNA Sample Preparation Kit (Illumina, San Diego, CA, USA), according to protocols supplied by Fluidigm. Two hundred eighty‐five single‐cell libraries and control libraries were successfully collected and sequenced by either 100‐bp paired‐end on the Illumina HiSeq 2500 or 150‐bp paired‐end on the NovaSeq 6000. Quality metrics of single‐cell RNA‐seq data (except two samples for which a very low number of reads were obtained) were as follows: mean reads per cell, 3.96 million reads/cell; percentage of reads mapped to the genome, average 80.43%; total genes detected, 16,408 genes; mean detected genes per cell, 3854.13 genes/cell. These sequence data were deposited in DDBJ Sequence Read Archive under the accession number DRA011310 and DRA011348.