characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-11-10T00:30:34Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-11-10T00:30:34Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112194193
characteristics.characteristic: External Id
characteristics.text: Institute of Physiological Chemistry, University of Ulm
characteristics.characteristic: INSDC center name
characteristics.text: 2023-11-10T00:30:34Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-11-10T00:30:34Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14305270
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12433:KNeC1
characteristics.characteristic: Submitter Id
characteristics.text: 10
characteristics.unit: month
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: Cryopreserved tissue was sectioned and placed in poly-L-lysine-coated membrane slides and air-dried for 5 minutes. Then, slides were incubated serially in 70% ethanol for 3 minutes, distilled water for 30 seconds, hemalum (Merck #109249, diluted 1:3 in distilled water) for one minute, distilled water for 30 seconds, 70% ethanol for 30 seconds, 90% ethanol for 30 seconds, 100% ethanol for 30 seconds and air-dried. Lesions were then excised and captured using the PALM Microbeam Rel. 4.9 with RoboMover microscope (Zeiss) and the PALMRobo v 4.9 software. Ethanol dilutions for the LCM preparation were stored at -20oC. RNAse-free water for the LCM preparation was stored at 4oC. RNA from isolated lesions was extracted with RNeasy Micro Kit (QIAGEN #74004) according to its standard protocol. RNAse-free water was used in all steps (including dilution of ethanol) to avoid RNA degradation. The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligoattached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. The products are then purified and enriched with PCR amplification. We then quantified the PCR yield by Qubit and pooled samples together to make a single strand DNA circle (ssDNA circle), which gave the final library.
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: intraepithelial neoplasia
characteristics.characteristic: disease
characteristics.text: Pdx1-Cre/LSL-KRASG12D/NEMOfloc
characteristics.characteristic: genotype
characteristics.text: T12
characteristics.characteristic: individual
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: pancreas
characteristics.characteristic: organism part
characteristics.text: precancerous lesion
characteristics.characteristic: sampling site
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: female
characteristics.characteristic: sex
characteristics.text: C57BL/6J
characteristics.characteristic: strain
characteristics.text: KNeC1
characteristics.characteristic: title