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Design: QuantSeq and RNAseq libraries to study developmental alternative polyadenylation
Submitted by: Institute of Molecular Life Sciences and Swiss Institute of Bioinformatics (Institute of Molecular Life Sciences and Swiss Ins)
Study: QuantSeq and RNAseq libraries to study developmental alternative polyadenylation
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QuantSeq-Rev method to generate highly strand-specific next-generation sequencing (NGS) libraries enabling transcript quantification and identification of the 3'end of polyadenylated RNAs
Name: 20151027_LUHMGU5_e39_s
Instrument: Illumina HiSeq 2500
Strategy: OTHER
Selection: RT-PCR
Layout: SINGLE
Construction protocol: 3 µg of RNA was treated with TURBO DNase, followed by RNeasy Minelute RNA cleanup kit and RNA quaility was confirmed using Agilent 2100 Bioanalyzer with RNA Pico 6000 kit (RIN values >8). 0.5 µg of RNA was used for Lexogen QuantSeq-REV libraries. cDNA libraries were prepared according to Lexogen QuantSeq Rev method manufacturer's protocol, using a poly(T) primer for reverse transcription. The library was sequenced using Illumina HiSeq, producing 60 nt single-end reads and 10 nt index reads H9 human ESCs (H9 line) were cultured in mTeSR1 medium on Matrigel-coated plates. Colonies were passaged for maintanence by Gentle Cell Dissociation Reagent We used shRNA targeting human TDP-43 (GAAACACAAGTGAAAGTAA) or a control targeting firefly luciferase (CGTACGCGGAATACTTCGA) driven by the H1 promoter in the vector FUW coexpressing TagRFP both for transfection and transduction, while maintaining hESC in pluripotency or differentiation growth conditions. Lexogen 3'-end QuandSeq Reverse
Experiment attributes:
Experimental Factor: undifferentiated human embryonic stem cell: cell type
Experimental Factor: shRNA transduction targeting TDP-43: RNA interference
Run# of Spots# of BasesSizePublished


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