Computational protocol: Coding and small non-coding transcriptional landscape of tuberous sclerosis complex cortical tubers: implications for pathophysiology and treatment

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Protocol publication

[…] Single cell RNA-Seq data sets produced by the Quake laboratory (Stanford University, CA), were retrieved from NCBI’s Gene Expression Omnibus (GEO) (accession no. GSE67835). Data was retrieved for four different cell types; neurons, oligodendrocytes, microglia and astrocytes, all from healthy human cortex. Three biological replicates for each cell type were used, giving 12 samples in total. Sequence reads were trimmed and filtered using FastQC v0.11.2 and Trimmomatic v0.33 as aforementioned. Paired-end reads were aligned to the human reference genome (GRCh38) with TopHat2 v2.0.13 using the default settings. Next, the number of reads that mapped to each gene in the genome was calculated using featureCounts from the SubRead package. The GRCh38 reference annotation file Gencode v21 was used as an input for featureCounts. Data analysis and manipulation was performed in R (version 3.2.4). The count matrix was normalized using the R package DESeq2. [...] miRNAs (miR-34a-5p, miR-34b-5p, miR-34c-5p, miR-302a-3p, miR-21-5p and the reference small nuclear RNAs, Rnu6B and Rnu44) expression was analyzed using Taqman micro RNA assays (Applied Biosystems, Foster City, CA). cDNA was generated using Taqman MicroRNA reverse transcription kit (Applied Biosystems, Foster City, CA) according to manufacturer’s instructions and the PCRs were run on a Roche Lightcycler 480 thermocycler (Roche Applied Science, Basel, Switzerland). Quantification of data was performed using the computer program LinRegPCR in which linear regression on the Log (fluorescence) per cycle number data is applied to determine the amplification efficiency per sample, . The starting concentration of each specific product was divided by the starting concentration of reference genes (geometric mean of Rnu6B and Rnu44 values) and this ratio was compared between groups.To evaluate expression of miRNA targets and inflammation-related genes, 2.5 µg of total RNA was reverse-transcribed into cDNA using oligodT primers. PCR primers (Eurogentec, Belgium) were designed using the Universal ProbeLibrary of Roche (https://www.roche-applied-science.com) on the basis of the reported cDNA sequences (Supplementary Table ). For each PCR, a mastermix was prepared on ice, containing per sample: 1 µl cDNA, 2.5 µl of 2x SensiFASTTM SYBR Green Reaction Mix (Bioline Inc, Taunton, MA, USA), 0.4 µM of both reverse and forward primers and the PCRs were run on a Roche Lightcycler 480 thermocycler (Roche Applied Science, Basel, Switzerland). Quantification of data was performed as described for the Taqman PCR and the starting concentration of each specific product was divided by the geometric mean of the starting concentration of reference genes (EF1A, C1orf43 and SNRPD3) and this ratio was compared between patient/control groups. […]

Pipeline specifications

Software tools FastQC, Trimmomatic, TopHat, Subread, DESeq2, LinRegPCR
Applications scRNA-seq analysis, qPCR
Organisms Mus musculus
Diseases Epilepsy, Movement Disorders, Tuberous Sclerosis, Genetic Diseases, Inborn
Chemicals Glutamic Acid, Sirolimus