5 protocols
AccessionNameType
P-MTAB-29807
data_generation
Images from the instrument were processed using the manufacturers SOLiD V4 ICS v4.0.2 and 5500 Series genetic Analyser ICS v1.2.1 software to generate sequence CSFASTA and quality QUAL files.
P-MTAB-29805
sequencing
Purification of poly(A) RNA NucleoTrap mRNA (User Manual, August 2010 / Rev. 04) (Macherey Nagel, Dueren, Deutschland). Following the SOLiD Total RNA-Seq Kit instructions (Life Technologies, protocol Part Number 4452437 Rev. B, July 2011), 100 ng of polyA RNAs were fragmented by incubation with RNAse III for 10 min in a 1e-5 l reaction volume containing 1X RNase III buffer supplied with the enzyme. Fragmented RNAs were then purified using the RiboMinus Concentration Module (Invitrogen, Carlsbad, Ca). The yield and size distribution of the fragmented RNA were assessed using the Quant-iT RNA assay kit with the Qubit fluorometer (Invitrogen, Carlsbad, Ca) and the RNA 6000 Pico Chip Kit with the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, Ca). A total of 50 ng of fragmented RNAs were hybridized and ligated with the SOLiD adaptator mix and reverse transcribed according to supplier instructions. The isolated cDNA were size selected around 200 pb using 2 rounds of Agencourt AMPure XP Reagent (Beckman Coulter). The cDNA were then amplified according to the SOLiD Total RNA-Seq Kit, Standard Input protocol for CLB-Ga, CLB-Re and NB1141 : 15 PCR cycles; Low Input protocol for NB1142 : 18 PCR cycles. The yield and size distribution of the cDNA were assessed using the Quant-iT HS DNA assay kit with the Qubit fluorometer (Invitrogen, Carlsbad, Ca) and the High Sensitivity DNA Assay Chip Kit on the Agilent 2100 Bioanalyzer. Templated beads were generated for sequencing by SOLiD EZ Bead System automates using standard manufacturers protocols. The four samples are multiplexed and a total of 500 pM with a titration of 0.4pM are used for emulsion, amplification and enrichment steps. The SOLiD V4 and 5500 systems generated paired-end sequences of 50 and 35 nucleotides for 5' and 3' end respectively. Multiplex samples were run on a two flowchips of the SOLiD V4 and one flowcell (6 lanes) of the 5500 SOLiD system.
P-MTAB-29806
sequencing
Purification of poly(A) RNA NucleoTrap mRNA (User Manual, August 2010 / Rev. 04) (Macherey Nagel, Dueren, Deutschland). Following the SOLiD Total RNA-Seq Kit instructions (Life Technologies, protocol Part Number 4452437 Rev. B, July 2011), 100 ng of polyA RNAs were fragmented by incubation with RNAse III for 10 min in a 1e-5 l reaction volume containing 1X RNase III buffer supplied with the enzyme. Fragmented RNAs were then purified using the RiboMinus Concentration Module (Invitrogen, Carlsbad, Ca). The yield and size distribution of the fragmented RNA were assessed using the Quant-iT RNA assay kit with the Qubit fluorometer (Invitrogen, Carlsbad, Ca) and the RNA 6000 Pico Chip Kit with the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, Ca). A total of 50 ng of fragmented RNAs were hybridized and ligated with the SOLiD adaptator mix and reverse transcribed according to supplier instructions. The isolated cDNA were size selected around 200 pb using 2 rounds of Agencourt AMPure XP Reagent (Beckman Coulter). The cDNA were then amplified according to the SOLiD Total RNA-Seq Kit, Standard Input protocol for CLB-Ga, CLB-Re and NB1141 : 15 PCR cycles; Low Input protocol for NB1142 : 18 PCR cycles. The yield and size distribution of the cDNA were assessed using the Quant-iT HS DNA assay kit with the Qubit fluorometer (Invitrogen, Carlsbad, Ca) and the High Sensitivity DNA Assay Chip Kit on the Agilent 2100 Bioanalyzer. Templated beads were generated for sequencing by SOLiD EZ Bead System automates using standard manufacturers protocols. The four samples are multiplexed and a total of 500 pM with a titration of 0.4pM are used for emulsion, amplification and enrichment steps. The SOLiD V4 and 5500 systems generated paired-end sequences of 50 and 35 nucleotides for 5' and 3' end respectively. Multiplex samples were run on a two flowchips of the SOLiD V4 and one flowcell (6 lanes) of the 5500 SOLiD system.
P-MTAB-29804
nucleic_acid_extraction
RNA extraction was done with Trizol as described in Fix et al. (2008) Genes Chromosomes Cancer. 47:819-34.
P-MTAB-29803
grow
Neuroblastoma cell lines CLB-GA and CLB-RE: described in Schleiermacher G,et al. Cancer Genet Cytogenet. (2003) 141:32-42. Primary tumors NB1141 and NB1142: fresh tumors samples obtained at surgery were placed in liquid nitrogen and stored frozen at -80 C or in liquid nitrogen before RNA extraction.