13 protocols
AccessionNameType
P-MTAB-29766
bioassay_data_transformation
Summarisation of the calls in GenomeStudio version 2011.1 module GeneExpression version 1.9.0. Background corrected, quantile normalised. Variance was stabilized using base-2 logarithmic transformation.
P-MTAB-29765
bioassay_data_transformation
Summarisation of the calls in GenomeStudio version 2011.1 module GeneExpression version 1.9.0. Background corrected, no normalisation.
P-MTAB-29764
image_acquisition
Arrays were scanned using Illumina BeadArray Reader (part of Illumina BeadStation 500), according to the manufacturer's protocol. Summarisation of the calls in GenomeStudio version 2011.1 module GeneExpression version 1.9.0. No normalisation, no background correction.
P-MTAB-29763
hybridization
750 ng of cRNA per hybridisation was hybridised according to the manufacturer's protocol (in Illumina Hybridization Oven).
P-MTAB-29762
labeling
200 ng of RNA was amplified and labelled using Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, USA) according to the manufacturer's protocol.
P-MTAB-29757
grow
Mouse myoblast cell line C2C12 were from ATCC (American Type Culture Collection, VA, USA, cat.no.# CRL-1772) and were cultured under standard conditions in DMEM supplemented with 20 % FBS (PAA, cat.no.# A15-102). In order to modify c-Myb expression, two constructs were prepared in retroviral vector PMSCV-IRES-eGFP kindly provided by Proffesor PS Zammit (King´s College London, London, UK) expressing gene of interest and eGFP from the same transcript. Retroviral vector expressing c-Myb (exoMyb) constitutively overexpress c-Myb, retroviral vector expressing miR-150 (miR-150) downregulates expression of endogenous c-Myb and empty expression vector served as a control (PMSCV). Retroviruses were packaged in EcoPack2TM-293 cell line according to the manufacturer´s manual (Clontech). C2C12 cells were incubated with medium containing virus supplemented with 4 mg/ml polybrene for 24 hours. C2C12 cells were seeded at 4x103cells/cm2 in GM, after 24 hours infected with retroviruses for 24 hours and then cultivated for another 24 hours in GM. Cells were then detached from culture dishes with trypsin-ethylenediamine tetraacetic acid (EDTA). For cell sorting, cells were transferred to growth medium and eGFP-expressing cells were separated with BD Influx TM cell sorter (BD Biosciences, NJ, USA). 2x104 C2C12 cells expressing c-Myb+eGFP (exoMyb), miR-150+eGFP (miR-150) or eGFP (PMSCV) were collected. Cell were expanded in DMEM supplemented with 20 % FBS, subconfluent cell were either harvested (D0) for microarray analysis or differentiated in differentiation medium consisting of DMEM supplemented with 2 % heat inactivated horse serum (Gibco, cat.no.# 26050-088). After culturing for 24 hours (D24) and 72 hours (D72), cells were harvested for microarray analysis. Western bloting at the same time points proved expected pattern of c-Myb expression. In C2C12 cells expressing c-Myb-eGFP c-Myb is upregulated in all time points, miR-150 expression downregulated c-Myb, there are only traces of endogenous c-Myb in D0, and c-Myb is absent in D24 and D72. Vector has no effect on c-Myb: c-Myb is expressed in D0, traces are detected in D24 and no expression is detected in D72.
P-MTAB-29759
specified_biomaterial_action
Mouse myoblast cell line C2C12 were from ATCC (American Type Culture Collection, VA, USA, cat.no.# CRL-1772) and were cultured under standard conditions in DMEM supplemented with 20 % FBS (PAA, cat.no.# A15-102). In order to modify c-Myb expression, two constructs were prepared in retroviral vector PMSCV-IRES-eGFP kindly provided by Proffesor PS Zammit (King´s College London, London, UK) expressing gene of interest and eGFP from the same transcript. Retroviral vector expressing c-Myb (exoMyb) constitutively overexpress c-Myb, retroviral vector expressing miR-150 (miR-150) downregulates expression of endogenous c-Myb and empty expression vector served as a control (PMSCV). Retroviruses were packaged in EcoPack2TM-293 cell line according to the manufacturer´s manual (Clontech). C2C12 cells were incubated with medium containing virus supplemented with 4 ?g/ml polybrene for 24 hours. C2C12 cells were seeded at 4x103cells/cm2 in GM, after 24 hours infected with retroviruses for 24 hours and then cultivated for another 24 hours in GM. Cells were then detached from culture dishes with trypsin-ethylenediamine tetraacetic acid (EDTA). For cell sorting, cells were transferred to growth medium and eGFP-expressing cells were separated with BD Influx TM cell sorter (BD Biosciences, NJ, USA). 2x104 C2C12 cells expressing c-Myb+eGFP (exoMyb), miR-150+eGFP (miR-150) or eGFP (PMSCV) were collected. Cell were expanded in DMEM supplemented with 20 % FBS, subconfluent cell were induced to differentiate by culturing in in the differentiation medium consisting of DMEM supplemented with 2 % heat inactivated horse serum (Gibco, cat.no.# 26050-088). After culturing for 24 hours cells were harvested for microarray analysis. The described experiment was repeated three times to obtain independent triplicate samples from which RNA were prepared
P-MTAB-29754
specified_biomaterial_action
C2C12 cell were infected with retrovirus expressing cMyb+eGFP. (PMSCV-c-Myb-IRES-eGFP). Retroviral vector PMSCV-IRES-eGFP was kindly provided by Proffesor PS Zammit (King´s College London, London, UK).
P-MTAB-29761
nucleic_acid_extraction
Total RNA was isolated using the TriZol Reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. Quantity of the RNA was measured on NanoDrop ND-1000 (NanoDrop Technologies LLC, Wilmington, DE, USA). RNA integrity was assessed on Agilent 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA, USA). All RNA samples had RIN above 9.
P-MTAB-29755
specified_biomaterial_action
C2C12 cell were infected with retrovirus expressing miR-150+eGFP (PMSCV-miR-150-IRES-eGFP). Retroviral vector PMSCV-IRES-eGFP was kindly provided by Proffesor PS Zammit (King´s College London, London, UK).
P-MTAB-29758
specified_biomaterial_action
Mouse myoblast cell line C2C12 were from ATCC (American Type Culture Collection, VA, USA, cat.no.# CRL-1772) and were cultured under standard conditions in DMEM supplemented with 20 % FBS (PAA, cat.no.# A15-102). In order to modify c-Myb expression, two constructs were prepared in retroviral vector PMSCV-IRES-eGFP kindly provided by Proffesor PS Zammit (King´s College London, London, UK) expressing gene of interest and eGFP from the same transcript. Retroviral vector expressing c-Myb (exoMyb) constitutively overexpress c-Myb, retroviral vector expressing miR-150 (miR-150) downregulates expression of endogenous c-Myb and empty expression vector served as a control (PMSCV). Retroviruses were packaged in EcoPack2TM-293 cell line according to the manufacturer´s manual (Clontech). C2C12 cells were incubated with medium containing virus supplemented with 4 ?g/ml polybrene for 24 hours. C2C12 cells were seeded at 4x103cells/cm2 in GM, after 24 hours infected with retroviruses for 24 hours and then cultivated for another 24 hours in GM. Cells were then detached from culture dishes with trypsin-ethylenediamine tetraacetic acid (EDTA). For cell sorting, cells were transferred to growth medium and eGFP-expressing cells were separated with BD Influx TM cell sorter (BD Biosciences, NJ, USA). 2x104 C2C12 cells expressing c-Myb+eGFP (exoMyb), miR-150+eGFP (miR-150) or eGFP (PMSCV) were collected. Cell were expanded in DMEM supplemented with 20 % FBS, subconfluent cell were harvested for microarray analysis.The described experiment was repeated three times to obtain independent triplicate samples from which RNA were prepared
P-MTAB-29756
specified_biomaterial_action
C2C12 cell were infected with retrovirus expressing eGFP (PMSCV-IRES-eGFP). Retroviral vector PMSCV-IRES-eGFP was kindly provided by Proffesor PS Zammit (King´s College London, London, UK).
P-MTAB-29760
specified_biomaterial_action
Mouse myoblast cell line C2C12 were from ATCC (American Type Culture Collection, VA, USA, cat.no.# CRL-1772) and were cultured under standard conditions in DMEM supplemented with 20 % FBS (PAA, cat.no.# A15-102). In order to modify c-Myb expression, two constructs were prepared in retroviral vector PMSCV-IRES-eGFP kindly provided by Proffesor PS Zammit (King´s College London, London, UK) expressing gene of interest and eGFP from the same transcript. Retroviral vector expressing c-Myb (exoMyb) constitutively overexpress c-Myb, retroviral vector expressing miR-150 (miR-150) downregulates expression of endogenous c-Myb and empty expression vector served as a control (PMSCV). Retroviruses were packaged in EcoPack2TM-293 cell line according to the manufacturer´s manual (Clontech). C2C12 cells were incubated with medium containing virus supplemented with 4 ?g/ml polybrene for 24 hours. C2C12 cells were seeded at 4x103cells/cm2 in GM, after 24 hours infected with retroviruses for 24 hours and then cultivated for another 24 hours in GM. Cells were then detached from culture dishes with trypsin-ethylenediamine tetraacetic acid (EDTA). For cell sorting, cells were transferred to growth medium and eGFP-expressing cells were separated with BD Influx TM cell sorter (BD Biosciences, NJ, USA). 2x104 C2C12 cells expressing c-Myb+eGFP (exoMyb), miR-150+eGFP (miR-150) or eGFP (PMSCV) were collected. Cell were expanded in DMEM supplemented with 20 % FBS, subconfluent cell were induced to differentiate by culturing in the differentiation medium consisting of DMEM supplemented with 2 % heat inactivated horse serum (Gibco, cat.no.# 26050-088). After culturing for 72 hours cells were harvested for microarray analysis.The described experiment was repeated three times to obtain independent triplicate samples from which RNA were prepared.