Homo sapiens
X43yr_E4__F Protocols: For the direct conversion of human fibroblasts into induced neurons (iN), cells were lentivirally transduced to express rtTA and a 2A-peptide-linked transcript coding for the pro-neuronal transcription factors Ngn2 and Ascl1, resulting in transgenic, but silent and expandable, AN fibroblasts. For iN conversion, cells were transferred to conversion media containing doxycycline and small molecular enhancers (also see Ladewig et al., Nat Methods 2012). Under these conditions, fibroblasts underwent marked morphological changes into neuronal cells, with typical neuronal morphologies appearing around week 2 of conversion. RNA was collected for RNA-Seq on various time points during the iN conversion process. To analyze the transcriptome of iNs via RNA-Seq, neuronal RNA needs to be pure, with minimal fibroblast contamination that might confound analysis. As live iNs express PSA-NCAM on the cell surface, we established a FACS-based protocol for purification of neurons using a PSA-NCAM antibody. Pure iN RNA for RNA-Seq was isolated by sorting directly into Trizol-LS reagent. All other fibroblast and iPSC RNA samples were directly collected from adherent mono-cultures. Trizol-LS and RNeasy mini TruSeq stranded mRNA (Illumina)
ENA-FIRST-PUBLIC
2015-09-14T17:01:06Z
ENA-LAST-UPDATE
2018-03-08T20:29:20Z
External Id
SAMEA3110457
INSDC center name
Laboratory of Genetics - Gage The Salk Institute for Biological Studies
INSDC first public
2015-09-14T17:01:06Z
INSDC last update
2018-03-08T20:29:20Z
INSDC status
public
Submitter Id
E-MTAB-3037:Sample 14
age
43 year
broker name
ArrayExpress
cell type
Primary human fibroblasts
common name
human
sample name
E-MTAB-3037:Sample 14
scientific_name
Homo sapiens