title: Sample 1
organism: Homo sapiens
description: Protocols: DMEM with 10% FBS Oligo (dT) are used to isolate mRNA(for eukaryotes) or by removing rRNAs from the total RNA (for prokaryotes). Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. Then cDNA is synthesized using the mRNA fragments as templates. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments are connected with adapters. After agarose gel electrophoresis, the suitable fragments are selected for the PCR amplification as templates. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used in quantification and qualification of the sample library. At last, the library could be sequenced using Illumina HiSeqTM 2000 or other sequencer when necessary. Total RNA of cells was extracted by Trizol (Invitrogen) The RNA fragments were then copied into first strand cDNA using reverse transcriptase and random hexamer primers followed by second strand cDNA synthesis using DNA Polymerase I and RNaseH. The short cDNA fragments were finally prepared for sequencing on the Illumina Genome Analyzer using reagents provided in the Illumina Genomic DNA Sequencing Sample Prep Kit. The cDNA fragments were end-repaired using T4 DNA polymerase and Klenow DNA polymerase and a single adenine nucleotide base was added to the cDNA fragments by using 3prime to 5prime exonuclease. This was followed by ligation of Illumina adaptors, gel purification of fragments of approximately 200 bases in size and finally PCR enrichment for 15 cycles. The concentrations of the libraries were measured using Invitrogen Qubit fluorometer and the size and purity were determined using the Agilent DNA 1000 Kit on Agilent 2100 Bioanalyzer. The 89 libraries were diluted in buffer EB from QIAGEN to 10 nM, denaturated with 2 N NaOH to a final DNA concentration of 1.0 nM, diluted to 4 pM with pre-chilled hybridization buffer from Illumina and loaded in individual lanes into a 1.0 mm flowcell together with a single lane of a 2 pM PhiX control library from Illumina.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2016-01-15T17:02:18Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-09T02:45:04Z
attributes.tag: External Id
attributes.value: SAMEA3474039
attributes.tag: INSDC center name
attributes.value: Tianjin Medical University Cancer Institute and Hospital
attributes.tag: INSDC first public
attributes.value: 2016-01-15T17:02:18Z
attributes.tag: INSDC last update
attributes.value: 2018-03-09T02:45:04Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-3710:Sample 1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: Panc1
attributes.tag: common name
attributes.value: human
attributes.tag: genetic modification
attributes.value: lentiviral transduction
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: sample name
attributes.value: E-MTAB-3710:Sample 1
attributes.tag: scientific_name
attributes.value: Homo sapiens