Illumina HiSeq 2000 paired end 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). Hepatocytes were isolated through classical collagenase perfusion of the tissue. More details can be found here: Li AP, Roque MA, Beck DJ, Kaminski DL. Isolation and culturing of hepatocytes from human livers. J Tissue Cult Meth. 1992;14:139–146. 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
normal
Experimental Factor: genotype
wild type alpha-1-antitrypsin