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CODING AND SMALL NON-CODING TRANSCRIPTIONAL LANDSCAPE OF TSC
the module membership measure, k34, 35. Significantly different miRNA expression indices between TSC and control patients were interrogated for their predicted interations with module-specific genes by means of the miR-Walk atlas, specifying the miR-Walk algo- rithm, of gene-miRNA-target interactions36. We selected for human species annotations and considered 3’UTR interactions as well as a minimum seed length equating to 7. All other parameters were default. Correlation analysis between miRNA profiles and module eigengenes was performed by Perason’s method. Significant correlations were demar- cated by p < 0.05.
Single cell RNA-seq analysis
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)37. 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 settings26. Next, the number of reads that mapped to each gene in the genome was calculated using featureCounts from the SubRead package38. The GRCh38 reference annotation file Gencode v21 was used as an input for featureCounts28. Data analysis and manipulation was performed in R (version 3.2.4). The count matrix was normalized using the R package DESeq239.
DNA analysis
TSC1 and TSC2 mutation analysis was performed by sequence analysis of all coding exons and exon/intron boundaries. Mutations are described according to HGVS nomenclature (Accession number NM_000548.3)40.
In situ hybridization
Insituhybridization(ISH)formiR34a-5pandmiR34b-5pwereperformedon5μmthickFFPE tissue using 5’ - 3’ double digoxygenin (DIG)-labeled probes as described previously41, 42. The probe sequences used were: miR34b-5p: 5’ DIG-AugGcaGugGagTuaGugAuuG-DIG;from Ribotask ApS (Odense, Denmark) and miR34a5p: 5’ DIG- AcaAccAgcTaaGacAcuGccA- DIG (Exiqon A/s, Vedbaek, Denmark) (capital letter = LNA modification, small letter = 2-o-methyl modification). Briefly, after the sections were deparaffinized and heat-treated to undo protein crosslinks (10 min at 120oC in a pressure cooker), the probes were hybridized at 56°C for 1 h. The hybridization was detected with an alkaline phosphatase (AP)-labeled anti-DIG antibody (Roche Applied Science, Basel, Switzerland). NBT (nitro- blue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt) was used as chromogenic substrate for AP. Negative controls sections were without probes and without primary antibody. For the double-staining, combining immunohis- tochemistry with ISH, the sections were first processed for ISH and then processed for immunohistochemistry with glial fibrillary acidic protein (GFAP, astrocyte marker; monoclonal mouse, Sigma, St. Louis, Mo, USA; 1:4000), NeuN (neuronal nuclear pro- tein; mouse clone MAB377; Chemicon, Temecula, CA, USA; 1:2000), and HLA-DP/DQ/
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