Comment[GEAAccession]	E-GEAD-616
MAGE-TAB Version	1.1
Investigation Title	Comparison of mock and RALB overexpressed iPSC-derived megakaryocytes
Experiment Description	Comparison of mock and RALB overexpressed iPSC-derived megakaryocytes. Lentivial-mediated RALB overexpression were performed in imMKCLs.
Experimental Design	genetic modification design
Experimental Factor Name	genetic_modification
Experimental Factor Type	genetic_modification
Person Last Name	Chen	Takayama
Person First Name	Si Jing	Naoya
Person Affiliation	Innovation regenerative medicine, Chiba University	
Person Roles	submitter	
Public Release Date	2024-02-23
Protocol Name	P-GEAD-1828	P-GEAD-1829	P-GEAD-1830	P-GEAD-1831	P-GEAD-1832
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid library construction protocol	nucleic acid sequencing protocol	normalization data transformation protocol
Protocol Description	The imMKCLs were cultured as described before.The full-length coding sequences of human CUX1 and RALB were cloned into the lentiviral vector CS2-Ubic-IG-GFP. Lentiviral production using a 293T system was described previously.	Total RNA was extracted using the microRNeasy Micro Kit according to the manufacturer guidelines. Briefly, cell lysates were prepared by adding the buffer RLT. The cell lysates were centrifuged to collect the supernatant. Ethanol was added and mixed properly. The lysates were collected into the spin column and centrifuged at 8,000 g, and the flow through was discarded. The column was washed with washing buffer (RW1) followed by a DNase treatment.	Up to 10 ng of total RNA was used for the cDNA synthesis using a SMART Seq v4 Ultra Low Input RNA Kit for sequencing (Takara Bio). cDNA was fragmented using an S220 Focused Ultrasonicator (Covaris, Woburn, MA, USA). The cDNA library was then generated using a NEBNext Ultra DNA Library Prep Kit for Illumina (New England BioLabs, Beverly, MA, USA). Finally, the NEBnext library size was estimated using a bioanalyzer with an Agilent High Sensitivity DNA Kit.	Sequencing was performed using a NextSeq 500 (Illumina) platform with a single-read sequencing length of 60 bp.	TopHat (version 2.1.1) was used to map to the reference genome (UCSC/hg19) with annotation data from iGenomes (Illumina). Gene expression levels were quantified using Cuffdiff (Cufflinks version 2.2.1) and expressed as fragments per kilobase of exon per million mapped sequence reads (FPKM).
SDRF File	E-GEAD-616.sdrf.txt
Comment[AEExperimentType]	RNA-seq of coding RNA
Comment[BioProject]	PRJDB15883
Comment[Last Update Date]	2024-02-23