Illumina HiSeq 2000 sequencing; RNA-seq of hepatocytes obtained through step-wise differentiation of hIPSCs from a patient with A1AT deficiency and its point mutation-corrected isogenic hIPSC line. Comparison to primary hepatocytes from a healthy donor and an A1AT-deficient patient.
RNA-seq of hepatocytes obtained through step-wise differentiation of hIPSCs from a patient with A1AT deficiency and its point mutation-corrected isogenic hIPSC line. Comparison to primary hepatocytes from a healthy donor and an A1AT-deficient patient.
Cells in a 12-well plate were washed once with PBS and then lysed with 350uL/well of RNA lysis buffer provided in the RNA extraction kit (Sigma, Cat No. RTN350-1KT). hIPSC-derived hepatocytes were differentiated to day 35 as outlined in the publication of Hannan et al., Nature Protocols, 2013. More details on the derivation of the hiPSC lines can be found in the protocol text from this publication: Yusa K, Rashid ST, Strick-Marchand H, Varela I, Liu PQ, Paschon DE, Miranda E, Ordóñez A, Hannan NR, Rouhani FJ, Darche S, Alexander G, Marciniak SJ, Fusaki N, Hasegawa M, Holmes MC, Di Santo JP, Lomas DA, Bradley A, Vallier L. Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells.  Nature. 2011 Oct 12;478(7369):391-4. The correction of the mutation was done by a combination of zinc finger nucleases and piggyBac. The protocol for RNA extraction followed the manufacturer's manual (Sigma, Cat No. RTN350-1KT). Numerical assessment of the integrity of RNA samples on the Bioanalyzer (Agilent Technologies) revealed very good quality of all samples submitted (RNA integrity numbers = 10). 550ng of total RNA was used for library preparation with the Illumina TruSeq RNA Samples Preparation V2 assay. Total RNA underwent a poly-A-pull down, fragmentation into the desired length (~300bp) and cDNA synthesis through reverse transcription using random primers. cDNA went into end repair, adapter ligation, and a small PCR reaction. The completed samples were non-strand specific, barcoded RNA-seq library, which were checked for quality on the MiSeq (paired-end, 150bp sequencing).
Experimental Factor: disease
Alpha-1-antitrypsin deficiency
Experimental Factor: genotype
wild type alpha-1-antitrypsin; corrected in vitro