L3.6pl_G2m3
Protocols: CRISPR/Cas9 plasmids generation: 1) Donor plasmid construction. 750 bp (LAMC2 construct) of 5' homology arm (HA) and 3' homology arm were amplified from PDAC gDNA and cloned in pDONOR vector. LF2A-EGFP-BGHpA insertion cassette was generated by gene synthesis (Thermo Fisher) and cloned in the 5'HA3'HA previously engineered pDONOR vector. 2) sgRNA design. Small guide RNA was designed using the http://crispr.mit.edu web tool. To select for the most suitable sgRNAs, we applied the following criteria: i. localization of the sgRNA as near as possible to the desired site of insertion to maximize homologous recombination efficiency, ii. Cas9-mediated double strand breaks downstream of STOP codon to prevent NHEJ-induced indels in the ORF, iii. guide selected to anneal at the intersection between the 5' homology arm and 3' homology arm so that the donor plasmid is protected from Cas9 cut, iv. minimum off-target score and maximum Doench activity score. sgRNAs used to modify PDAC cells was designed as follows: CCTCAGTTGAGAAATATTTATGG. 3) px330-IRFP Cas9 plasmid construction. Px330 Cas9 plasmid was obtained from addgene (ref. 42230) and was modified by the introduction of a SV40promoter-IRFP expression cassette downstream of Cas9 by FseI - EcoRI. In addition, the BbsI site of IRFP was silenced by site-directed mutagenesis. SgRNAs were cloned in px330-IRFP. CRISPR/Cas9 Lamc2 targeted cells generation: 1) Nucleofection. For L3.6pl and PANC-1 cells nucleofection, one million of single-cell trypsinized PDAC cells were nucleofected with 3 µg of donor plasmid and 1 µg of px330-IRFP Cas9 corresponding plasmids using Lonza nucleofector kit V (VVCA-1003) and program A-32 in an Amaxa-II nucleofector following manufacturer protocol. 2) FACS strategy and generation of single cell-derived PDAC cells. Nucleofected cells were cultured in DMEM/RPMI complete medium for 48 hours. Then, we isolated cells that were IRFP positive by FACS and we cultured them in complete medium for 20 days we sorted the cells for EGFP to confirm the donor integration. We then selected the cell population positive and negative for the expression of EGFP (LAMC2-LF2A-EGFP) and we named them EGFP+ and EGFP-, respectively. Cells were seeded in a 96-well format to derive single-cell clones. CRISPR/Cas9 Lamc2 targeted tumor generation and RNA sequencing: 1) Tumor growth. All animal experiments have been approved by the local ministry (IACUC protocol #992/2017-PR) and were performed in the animal facility under pathogen-free conditions. Single-cell suspensions of 2.5x105 L3.6pl and PANC-1 clones (EGFP+ or EGFP-) cells were subcutaneously injected into 6-week-old nude CD1 male mice (Charles River Laboratories). Tumor take was monitored visually and by palpation bi-weekly. Tumor diameter and volume were calculated based on caliper measurements of tumor length and height using the formula: tumor volume = (length x width2)/2. Animals were considered to bear a tumor when the maximal tumor diameter was over 2 mm. The LAMC2-EGFP+ and EGFP- derived tumors were washed in HBSS, minced with a bladder and disrupted and homogenization via the Tissue Lyser by using 5mm stainless steel beads.The single cells were resuspended in Eurogold TRIFAST buffer (Euroclone) for RNA isolation. Total RNAs were extracted with Eurogold TRIFAST kit (Euroclone) according to the manufacturer's instructions. RNA was DNase-treated using the Turbo DNase Kit (Thermo Fisher) for 30 min at 37 °C and the reaction was stopped by adding the inactivation reagent from the kit and incubation for 5 min at 37 °C (Thermo Fisher). The pellet was air-dried, resuspended in 25 µL nuclease-free water and the RNA concentration determined by nanodrop and purity (RIN) by Bioanalyzer. TruSeq stranded mRNA libraries were run on the Illumina NextSeq 500. We generated 40 million reads per lane and paired-end reads from sequencing were trimmed from adaptors and quality checked by the sequencing service. Reads were aligned against the human reference sequence GRCh38 (hg38) from Genome Reference Consortium using BWA (Li 2009). Read counts per gene were determined using the feature Counts method from the Subread R package (Liao 2014) based on the gene information in the gtf available here ftp://ftp.ensembl.org/pub/release-94/gtf/homo_sapiens/Homo_sapiens.GRCh38.94.gtf.gz.
ENA first public
2022-04-11
ENA last update
2022-04-11
External Id
SAMEA13856698
INSDC center alias
Institute of Genetics and Biophysics 'Adriano Buzzati-Traverso'
INSDC center name
Institute of Genetics and Biophysics 'Adriano Buzzati-Traverso'
INSDC first public
2022-04-11T00:19:32Z
INSDC last update
2022-04-11T00:19:32Z
INSDC status
public
Submitter Id
E-MTAB-11597:L3.6pl_G2m3
broker name
ArrayExpress
cell line
L3.6pl
cell type
epithelial cell
common name
human
developmental stage
adult
disease
pancreatic adenosquamous carcinoma
genotype
CRISPR/Cas9 mediated LAMC2 labeling
growth condition
subcutaneously injected in CD1 male mice
immunophenotype
EGFP-negative
organism part
liver
sample name
E-MTAB-11597:L3.6pl_G2m3