Computational protocol: REST suppression mediates neural conversion of adult human fibroblasts via microRNA‐dependent and ‐independent pathways

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Protocol publication

[…] Fibroblasts were transduced with the different lentiviral vectors (pB.pA or pB.mir9/124.pA ± RESTi), and both untransduced fibroblasts and fibroblasts transduced only with REST shRNA were used as controls (CTR). Cells were collected 5 days after transduction. RNA was extracted using RNeasy mini kit (Qiagen) with DNase treatment and sent for RNA‐seq to UCLA Clinical Microarray Core. cDNA libraries were prepared using the KAPA Stranded mRNA‐Seq Kit from KAPAbiosystems. The 50‐bp single‐end reads from the Illumina HiSeq 2000 were mapped to the human genome assembly (GRCh38) using STAR (2.4.0j) (Dobin et al, ) with default parameters. mRNA expression was quantified using the subread package FeatureCounts (Liao et al, ) quantifying to NCBI annotation (GRCh38). Read counts were normalized to the total number of reads mapping to the genome. Clustering and differential expression analysis were done with DESeq2 (Love et al, ). Downstream analyses were performed using in‐house R and unix scripts. Gene ontology analysis was done with the Functional Annotation Tool of DAVID Bioinformatic Resources 6.7 (Huang et al, ). To get a list of uniquely up‐regulated genes in the gene ontology analysis (Figs I and D), BH‐corrected P‐values < 0.001 were used to get the genes strongly up‐regulated in one group (fetal fibroblasts + pB.pA in Fig I and pB.pA + RESTi in Fig D), while genes with P‐value < 0.05 in the other group (adult fibroblasts + pB.pA in Fig I and pB.mir9/124.pA in Fig D) were removed from the gene list. This ensured that no genes that showed a strong trend for up‐regulation were classified as “not up‐regulated”. For the principal component analysis (PCA), one of the pB.pA + RESTi triplicate clustered with the pB.pA group which is most likely due to lack of co‐expression of pB.pA and REST shRNA as they are delivered on separate vectors. This group was excluded from further analysis. […]

Pipeline specifications

Software tools STAR, Subread, DESeq2, DAVID
Application RNA-seq analysis
Organisms Homo sapiens
Diseases Alzheimer Disease, Parkinson Disease, Neurodegenerative Diseases