MiRNA-mediated direct conversion of fibroblasts into neurons was initiated by treating fibroblasts with lentiviral reprogramming cocktail of reverse tetracycline-controlled transactivator rtTA (Addgene, 66810) with 1) inducible pT-miR-NS, 2) pT-miR-9/9*-124 with shCTL, or 3) pT-miR-9/9*-124 with shPTBP2. The day following lentiviral transduction, media change was supplemented with doxycycline (Sigma-Aldrich, D9891). Three days following initial transduction, media was changed again and supplemented with doxycycline and antibiotics. Five days post transduction, cells were plated onto poly-l-ornithine (Sigma-Aldrich, P4957), fibronectin (Sigma-Aldrich, F4759), and laminin (Sigma-Aldrich, L2020) coated coverslips followed by full media switch to neuronal media (ScienCell, 1521) the following day supplemented with 200 M dibutyl-cyclic AMP (cAMP; Sigma-Aldrich, D0627), 1 mM valproic acid (VPA; Sigma Aldrich, P4543), 10 ng/mL human BDNF (PeproTech, 450-02), 10 ng/mL human NT-3 (Peprotech, 450-03), 1 M retinoic acid (RA; Sigma-Aldrich, R2625), RevitaCell supplement (Gibco, A2644501), and antibiotics. DOX was supplemented every 2 days while half media changes occurred every 4 days until day 14 when cells were collected for RNA extraction.
Growth protocol
Human adult fibroblasts (GM02171) obtained from NIGMS Coreill Institute were maintained high glucose Dulbecco’s Modified Eagle Medium (Gibco, 11960044) containing 10% FBS (Gibco, 10437028), MEM non-essential amino acids (Gibco, 11140050), sodium pyruvate (Gibco, 11360070), GlutaMAX (Gibco, 35050061), HEPES (Gibco, 15630080), penicillin-streptomycin (Gibco, 15130122), and 2-mercaptoethanol (Gibco, 21985023).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRIzol Reagent (Invitrogen, 15596026) in combination with RNeasy mini kit (Qiagen, 74104). RNA quality (RIN > 9.6) was determined with 2100 Bioanalyzer (Agilent).
Label
biotin
Label protocol
GeneChip WT PLUS reagent kit was used to prepare RNA samples to biotinylated cDNAs according to the manufacturer's instructions.
Hybridization protocol
Labelled cDNAs were hybridized onto the Human Clariom D array according to the manufacturer's instructions.
Scan protocol
Array was scanned using GeneChip Scanner 3000.
Data processing
Array data was analyzed using manufacturer’s software, Expression Console followed by Transcriptome Analysis Console (TAC) using RMA gene analysis setting.
