Homo sapiens
Protocols: HeLa cells were grown in RPMI1640 supplemented with 10% FCS, glutamine and penicillin/streptomycin. The cells were reverse transfected using RNAiMAX (ThermoFisher Scientific) and siRNAs targeting SF1 (L-012662-01-0020, Dharmacon) or non-targeting siRNAs (D-001810-10-20) as control. After 24 hours, cells were re-transfected using the forward transfection protocol and 48 hours later harvested for RNA and protein. Total RNA was isolated using Isol-RNA lysis reagent (5 PRIME). RNA purity, integrity and concentration were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc, USA). Only RNA with a RIN value of 8.0 or higher and a ratio (28s/18s) of above 1.8 was taken forward for sample preparation. Samples were processed for library constructions following the manufactory instructions (Illumina Tru Seq Stranded Total RNA sample preparation v2 Guide, Part #15031048 Rev.E October 2013 - “Low sample protocol”). In brief, 0.5 μg of total RNA from each tissue were depleted for cytoplasmic rRNA using the Ribo-Zero ribosomal reduction chemistry, chemically fragmented for 8 min at 94oC, and processed for 1st strand synthesis cDNA and then 2nd strand synthesis using dUTP instead of dTTP. After some purification steps, the cDNA was then end-repaired, purified, adenylated at the 3'-ends, and purified before adding the indexed adaptor sequences using the TruSeq Stranded LT Kit Index set A. Each library preparation was then enriched by 10 cycles of PCR, purified and finally validated in regards to size and concentration. For sizing, libraries were analyzed on the Agilent 2100 Bioanalyzer using a DNA 1000 kit from Agilent Technologies. The Libraries were quantified by qPCR using the KaPa Library quantification Kits (KaPa Biosystems, Cat KK4824).
ENA first public
2016-11-29
ENA last update
2016-06-27
External Id
SAMEA4051791
INSDC center alias
Department of Biochemistry and Molecular Biology University of Southern Denmark
INSDC center name
Department of Biochemistry and Molecular Biology University of Southern Denmark
INSDC first public
2016-11-29T17:01:13Z
INSDC last update
2016-06-27T17:40:18Z
INSDC status
public
Submitter Id
E-MTAB-4866:HeLa_SF1-KD_replB
broker name
ArrayExpress
common name
human
rna interference
SF1
sample name
E-MTAB-4866:HeLa_SF1-KD_replB
strain
HeLa