Bulk RNAseq on invadosomes vs whole cells

Invadosomes were microdissected from PFA fixed NIH-3T3 Src Lifeact-mRuby cells with a PALM type 4 (Zeiss) automated laser micro dissector.Isolation of the total RNA from each sample was performed with Direct-zol RNA miniprep (Zymo) following the supplier procedures. The concentration of RNA was measured by spectrophotometry (NanoDrop 2000, ThermoFisher) for each sample. RNA quality was checked using Lab Chip GX touch HT (PerKin Elmer) and RNA chip DNA 5K/RNA CZE (Perkin Elmer).cDNA libraries were synthesized using 500 ng of RNA from each sample with the Illumina Stranded mRNA Prep Ligation (Illumina) kit following the supplier procedures. Briefly, the first step consists on capturing mRNAs with magnetic beads targeting their poly-A tail. Then, mRNAs were fragmented, reverse transcribed into double strand cDNA and 3’ adenylated adapters and anchors were ligated to both ends. Finally, the cDNA library was amplified by PCR with a number of PCR cycles adapted to the start material quantity. Libraries quality was checked with Xmark chip on the Lab Chip GX touch HT (Perkin Elmer). After quantification of individual libraries by q-PCR (Roche LC480) using the NEBNext library quant kit (New England BioLabs), all the samples were pooled in an equimolar manner. The quality and quantity of the pool were checked by qPCR (Roche LC480) with the same kit and Lab Chip GX touch HT (Perkin Elmer) before sequencing. mRNA sequencing was performed using Nextseq 2000 Illumina (paired-end 2x100 bp) with a minimum of 30 million reads per sample in the PGTB facility (INRAE - Pierroton).