High sensitivity ngs analysis kit

An aliquot of RNA (12μL) was used for cDNA synthesis with Superscript II ® reverse transcriptase (Thermo Fisher, USA) and random primers. cDNA was analyzed by PCR with a single pair of primers to screen for the Δ12 variant by amplifying an ORF7a fragment of approximately 406 bp [17 (link)]. Amplicons were analyzed by capillary electrophoresis on a Fragment Analyzer 5200 system using the High Sensitivity NGS Analysis Kit (Agilent Technologies, USA). Amplicons were also subjected to Sanger sequencing in Macrogen (Korea).

Single-cell sequencing for the tabula muris project was previously described [17 (link)]. In brief, single cells were captured in droplet emulsions using the GemCode Single-Cell Instrument (10x Genomics), and scRNA-seq libraries were constructed as per the 10x Genomics protocol using GemCode Single-Cell 3′ Gel Bead and Library V2 Kit. The samples were diluted in PBS with 2% FBS to a concentration of 1.000 cells per μL. Cells were loaded in each channel with a target output of 5.000 cells per sample. All reactions were performed in the BioRad C1000 Touch Thermal cycler with 96-Deep Well Reaction Module. Amplified cDNA and final libraries were evaluated on a Fragment Analyzer using a High Sensitivity NGS Analysis Kit (Advanced Analytical). Equal volumes of 16 libraries were pooled for sequencing on the NovaSeq 6000 Sequencing System (Illumina).
Sequencing of Fzt:DU and C57BL/6NRj samples were conducted by Genewiz (GENEWIZ Germany GmbH, Leipzig, Germany). Similar to the tabula muris project, single nuclei were captured in droplet emulsions on the 10xGenomics system and sequenced on the NovaSeq 6000 Sequencing System (Illumina, San Diego, CA, USA). In contrast to the tabula muris sequencing, cells were loaded with a target output of 10,000 cells per sample and the snRNA-seq libraries were constructed using Library V3 chemistry.