title: 204_R1
organism: Mus musculus
description: Protocols: PDAC tumors were isolated, minced and processed for single cell suspension. Tissues were digested in 2 ml of Digestion Buffer composed by Collagenase IV 3% in RPMI 10% FBS + 1% P/S, DNAasi I 30U/ul, Hepes 10 mM. The cell suspension was incubated for 30 min at 37oC on a rocker. Then, the digestion was stopped adding 1 mL of RPMI 10% FBS + 1% P/S. The cells suspension was filtered through a 100 µm cell strainer and kept on ice for 4 minutes. SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian (Clontech Laboratories, Inc., A Takara Bio Company,California, USA) protocol total RNA samples (0.25–10 ng) were reverse-transcribed using random priming into double-stranded cDNA in the presence of a template switch oligo (TSO). When the reverse transcriptase reaches the 5' end of the RNA fragment, the enzyme's terminal transferase activity adds non-templated nucleotides to the 3' end of the cDNA. The TSO pairs with the added non-templated nucleotide, enabling the reverse transcriptase to continue replicating to the end of the oligonucleotide. This results in a cDNA fragment that contains sequences derived from the random priming oligo and the TSO. PCR amplification using primers binding to these sequences can now be performed. The PCR adds full-length Illumina adapters, including dual barcodes for multiplexing. Ribosomal cDNA is cleaved by ZapR in the presence of the mammalian-specific R-Probes. Remaining fragments are enriched with a second round of PCR amplification using primers designed to match Illumina adapters.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-07-07T00:23:42Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-07-07T00:23:42Z
attributes.tag: External Id
attributes.value: SAMEA112769166
attributes.tag: INSDC center alias
attributes.value: Istituto di Ricerche Farmacologiche \"Mario Negri\" IRCCS
attributes.tag: INSDC center name
attributes.value: Istituto di Ricerche Farmacologiche \"Mario Negri\" IRCCS
attributes.tag: INSDC first public
attributes.value: 2023-07-07T00:23:42Z
attributes.tag: INSDC last update
attributes.value: 2023-07-07T00:23:42Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-12746:204_R1
attributes.tag: age
attributes.value: 20
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_line
attributes.value: KPC
attributes.tag: cell_type
attributes.value: Epcam+; CD45-PDAC cells
attributes.tag: common name
attributes.value: house mouse
attributes.tag: disease
attributes.value: pancreatic cancer
attributes.tag: genotype
attributes.value: KHKC-/-
attributes.tag: growth condition
attributes.value: in vivo until neoplasm formation
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: pancreas
attributes.tag: sample name
attributes.value: E-MTAB-12746:204_R1
attributes.tag: sampling site
attributes.value: neoplasm
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: strain
attributes.value: C57BL/6