mixed sample
Protocols: Liver hepatocellular carcinomas cell lines HepG2 and Huh7 from human and Hepa1-6 and Hepa-1c1c7 from mouse were grown to 80% confluency in DMEM (Sigma) supplemented with 10% foetal bovine serum (FBS) and antibiotics (100 g/l penicillin and 100 g/ml streptomycin) at 37C and 5% CO2. Cells were fresh-frozen in liquid nitrogen. RNA extraction 1. Excise 25 mg liver tissue sample (on dry ice) into Precellys tubes CK28-R, bertin technology 2. Remove the sample tube from the dry ice and immediately pipet 700 ul QIAzol Lysis Reagent into each sample tube. 3. Homogenize immediately on the TissueLyser for 2x10sec 5000 with 10sec break 4. Place the sample tube containing the homogenate on the benchtop at room temperature (15-25C) for 5 min. This step promotes dissociation of nucleoprotein complexes. 5. Add 140 ul chloroform and shake it vigorously for 15s. Thorough mixing is important for subsequent phase separation. 6. Place the sample tube on the benchtop at room temperature for 2-3 min. 7. Centrifuge at >6000g (max. 9000g for Precellys tubes) for 15 min at 4C. After centrifugation, the sample separates into 3 phases: an upper, colorless, aqueous phase containing RNA; a white interphase enriched with DNA; and a lower, red, organic phase containing proteins. For tissues with an especially high fat content, an additional, clear phase may be visible below the red, organic phase. The volume of the aqueous phase should be approximately 350 ul. 8. Transfer the upper aqueous phase to a new reaction tube. 9. Add 1 Vol% of isopropanol and mix thoroughly. Incubate 15 min at RT 10. Centrifuge full-speed, 20 min, 4C. 11. Wash with 750 ul 75% EtOH 12. Centrifuge full-speed, 10 min, 4C 13. Allow pellet to air-dry 14. Dissolve pellet in 20-50 ul RNase-free water Step2, Turbo DNase treatment (Ambion AM1907) Volume 10ug Total RNA ~ 1/10th volume DNase buffer 10ul Turbo DNase enzyme 2ul Water to 100ul Incubate 37C 30 minutes (mix inactivation agent) 0.15 volume inactivation agent 15ul Ribosomal RNA of 5 ug of DNase-treated total RNA was removed using the RiboZero (Epicentre RZC110424) kit following manufacturer's instructions. First Strand cDNA synthesis was performed using the Invitrogen it (11917-010) following manufacturer's instructions. Second strand cDNA synthesis was done by using a 10mM dUTP mix (Promega U1335) (dATP, dCTP, dGTP, dUTP). Make up reaction on ice Volume cDNA from first Strand cDNA synthesis 50ul 5x FSS buffer, 4ul DTT 2ul, 5xSSS buffer 30ul DU/NTP mix 3ul, E.coli DNA ligase 1ul, E.Coli DNA polymerase 3ul, E.Coli Rnase H 1ul, Water 56ul, Incubate 16C for 2 hours, Add 2ul T4 DNA polymerase 5 minutes 16C. Qiagen column purification column Elute 50ul water or EB. Library was prepared following Illumina TruSeq RNA sample prep Low Throughput kit instructions with the exception to treat sample with 1ul Uracil N-glycosylase (UNG) 37C 15minutes (Applied Biosystems) before PCR amplification.
ENA-FIRST-PUBLIC
2016-04-18T17:01:17Z
ENA-LAST-UPDATE
2018-03-09T09:25:25Z
External Id
SAMEA3669189
INSDC center name
Cambridge Institute University of Cambridge Cancer Research UK
INSDC first public
2016-04-18T17:01:17Z
INSDC last update
2018-03-09T09:25:25Z
INSDC status
public
Submitter Id
E-MTAB-4052:HepG2_rep2_F
age
immortal cell line
broker name
ArrayExpress
cell line
HepG2
cell type
hepatocyte
common name
human
disease
hepatocellular carcinoma
organism part
liver cell line
sample name
E-MTAB-4052:HepG2_rep2_F
scientific_name
Homo sapiens
sex
male
component_organism
Homo sapiens
component_organism
Homo sapiens