GSM3684267: CARko_8; Mus musculus; RNA-Seq
Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell.
GEO
GSM3684267
GEO Accession
GSM3684267