GSM2668217: M1_p2_1; Mus musculus; RNA-Seq
Bone marrow of 5-week old male C57BL/6J wildtype mice purchased from Charles River or from in house breedings of C57BL/6J wildtype mice was isolated from femora and tibiae by flushing using 1x MojoSort™ Buffer (BioLegend®, 480017). Hematopoietic progenitors were enriched using the MojoSort™ Mouse Hematopoietic Progenitor Cell Isolation Kit (BioLegend®, 480004), stained and sorted according to different surface markers. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described18. 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. CEL-Seq2 (Hasimshony et al. 2016, Genome Biology, DOI: 10.1186/s13059-016-0938-8)
GEO Accession
GSM2668217