Kapa s library quantification kit

The SMARTer Ultra Low Input RNA Kit for Sequencing – v3 (Clontech Laboratories, #634851) was used to generate cDNA from a single cell following the manufacturers protocol. The cDNA was then visualized and quantified using the High Sensitivity DNA assay on a 2100 Bioanalyzer (Agilent Technologies, #5067-4626). Sequencing libraries were prepared with 2ng of cDNA using the Low Input Library Prep Kit (Clontech Laboratories, #634947) following the manufacturers protocol. Final libraries were validated via the High Sensitivity DNA assay on a 2100 Bioanalyzer (Agilent Technologies, 5067-4626) and quantified by qPCR using Kapa’s Library Quantification Kit (Kapa Biosystems, #KK4824) on the 7900HT (Applied Biosystems). Libraries were pooled prior to sequencing by 75 bp paired-end reads using reagents from the MiSeq 150 cycle kit v3 (Illumina, #MS-102-3001).

Sequencing and analysis by using Illumina HiSeq was performed at the Translational Genomics Research Institute, Arizona. Sequencing libraries were prepared with 250 ng of total RNA using Illumina's Truseq RNA Sample Preparation Kit v2 (Illumina, Inc.) following the manufacturer's protocol. In brief, poly-A containing mRNA molecules were purified using poly-T oligo attached magnetic beads. The mRNA was then thermally fragmented and converted to double-stranded cDNA. The cDNA fragments were end-repaired, a single “A” nucleotide was incorporated, sequencing adapters were ligated, and fragments were enriched with 15 cycles of PCR. Final PCR-enriched fragments were validated on a 2200 TapeStation (Agilent Technologies) and quantitated via qPCR using Kapa's Library Quantification Kit (Kapa Biosystems) on the QuantStudio 6 Flex Instrument (ThermoFisher). The final library was sequenced by 50 bp paired-end sequencing on a HiSeq 2500 (Illumina). Illumina BCL files were converted and demultiplexed (bcl2fastq 2.17). FASTQ files were trimmed of adapter sequences (CutAdapt 1.8.3) and aligned to rn5 (STAR 2.5). Aligned reads were summarized as gene-level counts (featureCounts 1.4.4). Sequencing and quality control reports were generated (FastQC 0.11.4 and Qualimap 2.1.3). The FASTQ files from the Illumina platform have been separately submitted to GEO under the accession number: GSE101798.