E-MTAB-15190:MPNST2 - ONT - PromethION
2025-08-27
ENA first public
ERC000011
ENA-CHECKLIST
GenomiqueENS, Institut de Biologie de l'ENS (IBENS), Département de biologie, École normale supérieure, CNRS, INSERM, Université PSL
INSDC center name
public
INSDC status
ERS24815724
SRA accession
E-MTAB-15190:MPNST2 - ONT - PromethION
Submitter Id
12.3
month
age
ArrayExpress
broker name
tumor cell
cell type
not collected
collection date
house mouse
common name
Protocols: As described in Lebrigand et al. 2020, we depleted the cDNA for variable extended (30–50%) cDNA that lacks poly(A) and poly(T) sequences. We re-amplified 10 ng of the 10x Genomics PCR product for  5 cycles with 5′-NNNAAGCAGTGGTATCAACGCAGAGTACAT-3′ and 5′ Biotine-AAAAACTACACGACGCTCTTCCGATCT 3′. After 0.6x SPRIselect purification to remove excess biotinylated primers, biotinylated cDNA (in 40 μl EB) is bound to 15 μl 1x SSPE washed Dynabeads™ M-270 Streptavidin beads (Thermo) resuspended in 10 μl 5x SSPE for 15 min at room temperature on a shaker. After two washes with 100 μl 1x SSPE and one wash with 100 μl EB, the beads are suspended in 100 μl 1x PCR mix and amplified for 9 cycles with the primers NNNAAGCAGTGGTATCAACGCAGAGTACAT and NNNCTACACGACGCTCTTCCGATCT to generate enough material for Nanopore sequencing library preparation. Amplified cDNA was purified with 0.6x SPRISelect  and Nanopore sequencing libraries were prepared with the Oxford Nanopore SQK- LSK-110[MOU2]  kit following the manufacturer’s instructions. All PCR amplifications for Nanopore library preparations were done with KapaHifi Hotstart polymerase (Roche Sequencing Solutions): initial denaturation, 3 min at 95 °C; cycles: 98 °C for 30 s, 64 °C for 30 s, 72 °C for 5 min; final elongation: 72 °C for 10 min, primer concentration was 1 μM.
description
primary MPNST
developmental stage
Prss56cre/+, R26mt/+, NF1flox/-
genotype
not collected
geographic location (country and/or sea)
Mouse ID 2022_26
individual
not applicable
isolate
ONT
library
Mus musculus
organism
Mus musculus
organism
tumor
organism part
Mus musculus
scientific_name
PromethION (long-reads)
sequencing platform
male
sex
C57BL/6J
strain
MPNST2 - ONT - PromethION
title