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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_e34_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 Primitive streak differentiation was induced by dissociation of colonies into single cells using accutase and subsequently seeding them on Matrige-coated 6-well plates containing mTESR1 medium  supplemented with ROCKi at 100% confluency. Next day cells were washed with PBS, and the medium was changed to RPMI1640 medium supplemented with L-Glutamine, B27 without insulin, 10µM ROCK inhibitor and 10µM CHIR99021. For trophoblast directed differentiation human ESCs were passaged using 0,25% Trypsin-EDTA and plated as single cells (105,000 cells/cm2) on MG coated plates in DMEM/F12 medium, supplemented with 20% KnockOut Serum Replacement, Glutamax, nonessential amino acids, beta-mercaptoethanol, and 50 ng/ml BMP4. Medium was changed daily. Lexogen 3'-end QuandSeq Reverse
Experiment attributes:
Experimental Factor: undifferentiated human embryonic stem cell: cell type
Experimental Factor: shRNA control targeting firefly luciferase: RNA interference
Run# of Spots# of BasesSizePublished


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