Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: (thymus MCs): Thymi were extracted, mechanically disrupted and enzymatically digested at 37oC for 3x15 minutes using 0.01% (w/v) Liberase TM (Roche) and 0.1% (w/v) DNase I (Sigma-Aldrich). Final digests were harvested, pooled and maintained at 4oC in FACS buffer (PBS, 0.1% (w/v) BSA, 0.02% (w/v) NaN3). Primary cells were then stained with monoclonal antibodies prior to being sorted by FACS directly in 300 ul of Trizol reagent. Sorted cells were sorted according to the cell surface phenotype Lin- SCA1+ (Lineage negative cocktail being CD45, CD2, CD4, CD11b, GR-1, B220, TER119, CD41, EpCAM, CD31). A minimum of 25 000 primary sorted cells were used per sample. Total RNA was extracted using TRIzol reagent (Invitrogen) and further purified using the RNeasy micro kit (Qiagen). (Bone MCs) Femurs, tibias and pelvis were collected, cleaned and washed 3 times in cold PBS. Using sharp surgical scissors, each bone was first cut longitudinally and then transversely to generate tiny fragments of ~1 mm2. Bone fragments were then washed 3 times in cold PBS and incubated with agitation (80 RPM) for 3x20 minutes at 37oC in the same enzymatic cocktail as above. Following each incubation, supernatants were added to HBSS medium (Invitrogen) supplemented with 2% (v/v) FBS, 10mM HEPES and 1% (v/v) penicillin-streptomycin. To further retrieve endosteal stromal cells, remaining bone fragments were gently crushed in supplemented HBSS medium using a pestle and mortar (5 x 50 gentle taps) and supernatants were pooled to previous ones. Red blood cell lysis was then performed on the resulting cell suspension prior to its filtration and resuspension in FACS buffer. Primary cells were then stained with monoclonal antibodies prior to being sorted by FACS directly in 300 ul of Trizol reagent. Sorted cells were sorted according to the cell surface phenotype Lin- SCA1+ (Lineage negative cocktail being CD45, CD2, CD4, CD11b, GR-1, B220, TER119, CD41, EpCAM, CD31). A minimum of 25 000 primary sorted cells were used per sample. Total RNA was extracted using TRIzol reagent (Invitrogen) and further purified using the RNeasy micro kit (Qiagen). (Skin MCs) Mouse trunk and dorsal skin (~12 cm2 / mouse) was aseptically dissected and incubated for 45 minutes in 0.01% (w/v) liberase. Dermis was then mechanically isolated and incubated with agitation (80 RPM) at 37oC for 2x30 minutes in the same enzymatic cocktail as above. Post-incubation, supernatants were filtered and pooled to 25 mL of PBS supplemented with 1% FBS and 5mM EDTA. Final cell suspension was then centrifuged and resuspended in FACS buffer. Primary cells were then stained with monoclonal antibodies prior to being sorted by FACS directly in 300 ul of Trizol reagent. Sorted cells were sorted according to the cell surface phenotype Lin- SCA1+ (Lineage negative cocktail being CD45, CD2, CD4, CD11b, GR-1, B220, TER119, CD41, EpCAM, CD31). A minimum of 25 000 primary sorted cells were used per sample. Total RNA was extracted using TRIzol reagent (Invitrogen) and further purified using the RNeasy micro kit (Qiagen). Transcriptome librairies were generated from total RNA using the TruSeq Stranded mRNA Library Prep Kit (Illumina) following the manufacturer’s protocol. Enrichment of RNA-seq stranded libraries with adapter molecules on both ends was done using 15 cycles of PCR amplification using the Illumina PCR mix and primers cocktail. Paired-end (2 x 100 bp) sequencing was peformed using the Illumina HiSeq 2000 running TruSeq v3 chemistry. Three RNA-seq librairies were sequenced per lane (8 lanes per flow cell).