Smarter ultra low rna kit

Full-length double-stranded cDNA was generated from 5 ng of total RNA and amplified using the SMARTer Ultra Low RNA Kit (Illumina, San Diego, CA, USA). Library preparation was performed from 10 ng of fragmented cDNA using the NEBNext Chip-Seq Library Prep protocol (New England BioLabs, Frankfurt am Main, Germany). Libraries were sequenced on an Illumina Hiseq2000 with 2 × 50-bp paired-end reads (n = 152).

Cells from the two donors (#42 and #123, from a previous study [3 (link)]) were TCR-activated as previously described, and maintained in 2 ml OpTmizer expansion medium with 5% FBS and100 U/ml IL-2 for 72h. Dead cells were removed by centrifugation on a 3 ml Percoll gradient at 800 x g for 10 minutes. Cells were washed, counted and resuspended in PBS. Cells were either used for bulk RNA-Seq or loaded to the Fluidigm C1 platform for single-cell capture and single-cell RNA-seq library generation according to the manufacturer’s protocol. Briefly, a cell suspension of approximately 300,000 cells/ml was introduced into the medium size chip (10–17 μm plate), suitable to capture cells of 10–17 ± 2 μm, and thus able to capture activated cells (usually ranging from 10 to 13 μm). After cell separation and capture, empty or debris-occupied wells were identified by microscope visualisation and discarded from subsequent analysis. cDNA libraries were then produced directly and automatically on the chip with Clontech SMARTer Ultra Low RNA kit for Illumina using manufacturer-provided protocols. Illumina libraries were constructed in 96-well plates using the Illumina Nextera XT DNA Sample Preparation kit according to a protocol supplied by Fluidigm and sequenced on HiSeq2500 machine (Illumina), with 50 bp paired-end, 14 libraries multiplexed per lane.