GSM2771865: UCK KO Rep3; Mus musculus; RNA-Seq
To isolate bone marrow HSPCs, single-cell suspensions were prepared as described above and red blood cells were lysed. Cells were incubated with c-Kit-biotin (1:50) for 30 min at 4 ºC. Cells were then washed twice with HBSS with 2% FBS, and incubated with anti-biotin microbeads (Miltenyi) and positively selected using a MACS stand and a MS column (Miltenyi) following the manufacturer’s protocol. Positively selected cells were then stained with a lineage marker cocktail in FITC (CD4, CD8a, CD11b, B220, Gr-1, Ter119), streptavidin PE, and Sca-1 PE-Cy7. HSPCs were defined as Lin- cKit+ Sca-1+, and sorted using the Sony SH800 cell sorter, and collected directly into TRIzol (Thermo Fisher Scientific). Using NuGEN Ovation RNA-Seq System v2, purified double-stranded cDNA was generated from 10 ng of total RNA and amplified using both 3’ poly (A) selection and random priming. 2 µg of each sample was sheared using the Covaris S2 focused-ultrasonicator following the manufacturer’s protocol to obtain a final library with insert size of 400 bp. The sheared samples were quantified using the NanoDrop ND-1000 spectrophotometer and Invitrogen Qubit 2.0 DNA quantitation assay. The fragment sizes were confirmed on the Agilent Bioanalyzer to verify proper shearing. A double-stranded DNA library was produced using Illumina TruSeq DNA library preparation system and the sequencing was run on a HiSeq 2500 system.