Smartseq ht ultra low input kit

CD45.2⁺NGFR(ICN1)⁺ cells were sorted from the spleens of chimera mice at median survival time-point (4 weeks post-transplantation). RNA was isolated from these cells using the TRIzol reagent and submitted to Azenta Life Sciences LLC (South Plainfield, NJ, USA) for library preparation and Ultra-low RNA sequencing. Total RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies). The SMARTSeq HT Ultra Low Input Kit was used for full-length cDNA synthesis and amplification (Clontech), and Illumina Nextera XT library was used for sequencing library preparation. Briefly, cDNA was fragmented, and adaptor was added using Transposase, followed by limited-cycle PCR to enrich and add index to the cDNA fragments. Sequencing libraries were validated using the Agilent TapeStation and quantified by using Qubit Fluorometer as well as by quantitative PCR (KAPA Biosystems). The sequencing libraries were multiplexed and clustered on a flowcell. After clustering, the flowcell was loaded on the Illumina NovaSeq 6000 instrument according to manufacturer’s instructions. The samples were sequenced using a 2×150 Paired End (PE) configuration.