Agilent high sensitivity d5000 screentape

We labeled each group of NALT cells separately with the Sample-Tags from the BD Mouse Immune Single-Cell Multiplexing Kit (BD Biosciences, San Jose, CA), described in “Single Cell Labeling with the BD Single-Cell Multiplexing Kits” protocol. We then proceeded to library preparation with a mixture of ∼6000 cells (3000 cells from each group). We prepared the whole transcriptome using the BD Rhapsody System following the BD “mRNA Whole Transcriptome Analysis (WTA) and Sample Tag Library Preparation Protocol”. We assessed the quality and quantity of the final library by Agilent 4200 TapeStation system using the Agilent High Sensitivity D5000 ScreenTape (Agilent Technologies, Santa Clara, CA) and a Qubit Fluorometer using the Qubit dsDNA HS Assay (Invitrogen, MA, USA), respectively. The final library was diluted to 3 nM concentration and the sequencing was performed used a HiSeq PE150 sequencer (Illumina, San Diego, CA).

Single-cell RNA-seq libraries were generated using the Chromium Next GEM Single Cell v 3.1 (10X Genomics, US). Single-cell suspensions were diluted to a density of 1000 cells/μL in 1x PBS with 0.04% BSA and added to the real-time polymerase chain reaction (RT-PCR) master mix to target ∼8000 cells. The mix was loaded together with Single Cell v3.1 gel beads and partitioning oil into a Next GEM Chip G according to the manufacturer’s instructions. The cDNA molecules were amplified, dual indexed and pooled, and followed by library construction according to the manufacturer’s (10x genomics, US) instructions. Sequencing libraries were quantified by Qubit dsDNA High sensitivity kit and the size profiles of the pre-amplified cDNA and libraries were determined using the Agilent High Sensitivity D5000 ScreenTape. All single-cell libraries were sequenced with a customized paired-end format with dual indexing (28/10/90-bp for v3.1libraries) as recommended by 10X Genomics. These libraries were sequenced on a Novaseq 6000 system using the S1 flow cell (Illumina, US).