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Protocol publication

[…] ed from the selected cells using the SMARTer Ultra Low RNA kit designed for the C1 system (Clontech). mRNA libraries were constructed using the Nextera XT kit (Illumina) according to the manufacturer’s protocol. The libraries were sequenced on an Illumina MiSeqv3 with 75bp paired-end reads to a depth of 19-22 million reads per lane. Target sequencing depth for each library was 200K reads. The RNA-seq data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO series accession number GSE108849 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE108849)., Processing of RNA-seq data: Paired-end 75bp reads were mapped to the UCSC human transcriptome (hg19) using Bowtie2 (version 2.2.4) and Tophat (version 2.0.9). Gene expression levels were calculated using Cuffquant (Cufflinks version 2.2.1 with parameters -u -max-bundle-frags 10000000) and Cuffnorm (Cufflinks version 2.2.1). FPKM values as estimated by Cufflinks were added a value of 1 (to avoid zeros) and log2 transformed. We removed nine single-cell samples with low read counts (< 50K) and sub-sampled two single-cell samples sequenced as population controls with high read counts (> 1.5M) (random sub-sampling, 10% of total reads). 11 single-cell samples were excluded as outliers. We excluded lowly expressed genes (average log2 (FPKM) < 1.5) from further analysis. The remaining 543 single-cell samples met the requirement of expressing at least 2,000 of these remaining 5,196 genes. For each individual, the number of single-cells used in the analysis and the average number of reads for those single-cells is summarized in . The total […]

Pipeline specifications

Software tools Bowtie2, TopHat, Cufflinks
Databases GEO
Organisms Homo sapiens