Illumina HiSeq 4000 paired end sequencing; GSM3381002: iPS wt 5j A1 [JCZ_1]; Homo sapiens; RNA-Seq

iPS wt 5j A1 [JCZ_1]

Purpose: Here we describe the modulation of a gene expression program involved in cell fate. Methods: We depleted U2AF1 in human induced pluripotent stem cells (hiPSCs) to the level found in differentiated cells using an inducible shRNA system, followed by high-throughput RNAseq, revealing a gene expression program involved in cell fate determination. Results: Approximately 85% of the total raw reads were mapped to the human genome sequence (GRCh37), giving an average of 200 million human reads per sample for total RNA and 15 million human reads per sample for small RNA libraries. Conclusions: Our results show that transcriptional control of gene expression in hiPSCs can be set by the CSF U2AF1, establishing a direct link between transcription and AS during cell fate determination. Overall design: hiPSCs were differentiated into the three germ layers following the described protocol in the study (Gifford et al., 2013).

Transcription-dependent control of stem cell self-renewal and differentiation by the splicing factor U2AF1

Submitted by Gene Expression Omnibus on 04-DEC-2018

Total RNA extraction was performed according to the Qiagen miRNeasy kit. RNA concentrations were determined using Qubit fluorometric quantification and purity by the RNA 6000 Bioanalyzer kit (Agilent). RNA-Seq libraries were constructed with the TruSeq stranded mRNA sample preparation (Low throughput protocol) kit and TruSeq Small RNA protocol from Illumina. The first step of the pipeline is transcripts purification by depleting abundant ribosomal RNAs with specific RiboZero kit. 3 μg of high quality total RNA was eluted in 10 μL RNase-free water for the construction of the libraries. All samples were supplied at the same concentration of 300 ng/μL and were DNase treated. The Illumina TruSeq Small RNA protocol is optimized to target small RNAs with 5'-monophosphate. This option is required for samples with unknown 5'-phosphorylation status. The final cDNA libraries were validated with a Fragment Analyzer (Advanced Analytical, Ankeny, IA) and quantified with a KAPA qPCR kit (Kapa Biosystems, Wilmington, MA). Having passed quality control, the libraries were sequenced on Illumina HiSeq 4000.

Transcription-dependent control of stem cell self-renewal and differentiation by the splicing factor U2AF1

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