E-MTAB-6529:gmPNCTR_s
Illumina HiSeq 2500 sequencing; RNA-seq analysis of HeLa cells treated with GapmeR antisense oligonucleotides
RNA-seq analysis of HeLa cells treated with GapmeR antisense oligonucleotides
gmPNCTR_s
HeLa cells were plated overnight in a 12-well plate at 2x10^5/well in 1 ml/well of Gibco DMEM, high glucose DMEM medium containing GlutaMAX and pyruvate (Thermo Fisher Scientific, cat# 31966021) and additionally supplemented with 10% FBS (Hyclone; cat# SV30180.03) at +37C, 5% CO2. Next morning, each well was transfected with 25 pmol of an appropriate GapmeR oligonucleotide (Exiqon) mixed with 3 µl of Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific; cat# 13778150) and 100 µl of OPTI-MEM (Thermo Fisher Scientific; cat# 31985070), as recommended, and the incubation was continued for 24 hours prior to RNA extraction. Transfected HeLa cells were lysed with TRIzol (Thermo Fisher Scientific; cat# 15596026) and the total RNAs were isolated using PureLink RNA Mini Kit (Thermo Fisher Scientific; cat# 12183018A) with on-column PureLink DNAse (Thermo Fisher Scientific; cat# 12185010) treatment, according to the manufacturer's recommendations. The RNAs were eluted in nuclease-free water, QC'd (Bioanalyzer RIN score 9.8) and hybridized with oligo(dT) magnetic beads to isolate the poly(A) RNA fraction used for subsequent library preparation steps. Stranded mRNA sequencing libraries were prepared using the TruSeq Stranded mRNA Library Preparation Kit (Illumina cat## RS-122-2101 and RS-122-2102) as recommended.  Purified libraries were qualified on an Agilent Technologies 2200 TapeStation using a D1000 ScreenTape assay (cat## 5067-5582 and 5067-5583).  The molarity of adapter-modified molecules was defined by quantitative PCR using the Kapa Library Quant Kit (Kapa Biosystems; cat# KK4824).  Individual libraries were normalized to 10 nM and equal volumes were pooled in preparation for Illumina sequence analysis.
Centre for Developmental Neurobiology, King's College London
E-MTAB-6529:gmPNCTR_s
Experimental Factor: compound
GapmeR_PNCTR
Experimental Factor: dose
25