Illumina HiSeq 2000 paired end sequencing; Polysome profiling analysis of myeloid progenitors of control mice or mice conditionally deficient for Elp3 in the hematopoietic system
Polysome profiling analysis of myeloid progenitors of control mice or mice conditionally deficient for Elp3 in the hematopoietic system
Bone marrow cells were isolated by flushing femurs, tibias, ilia and humerus with PBS. Cells were filtered through a 70µM cell strainer to obtain single cell suspensions and cell number was assessed by counting with a hemocytometer. Cell suspensions were first enriched for lineage-negative cells by MACS using Mouse Lineage cell depletion kit (Miltenyi Biotec) before staining for cell-surface antigens. Cells were stained for 30 minutes using the following rat anti-mouse monoclonal antibodies: Lineage cocktail (145-2C11, RA3-6B2, RB6-8C5, M1/70 and TER-119)-PE, CD34 (RAM34) -FITC, CD150 (Q8-480)-BV421, CD150 (mShad150) - PerCP-eFluor710, CD48 (HM48-1) -APC-Cy7, CD127 (SB/199) -V450. All reagents were purchased from ThermoFisher Scientific, BD Bioscience or BioLegend. For polysome profiling analysis, 1-2x10^6 CD127- MyPs pooled from 5 Elp3fl/fl and 10 Elp3HscKO mice were FACS-sorted in biological triplicate on a FACS Aria 2 (BD Bioscience) Pellets were lyzed in hypotonic buffer as in (Liang et al., 2017, https://doi.org/10.1093/nar/gkx940) and 10% of each sample, corresponding to total RNA, were directly lyzed in Trizol and snap-frozen. The remaining cells were further loaded in a non-linear gradient as described in (Liang et al., 2017). Fractions corresponding to heavy polysomes were pooled, lyzed in Trizol and snap-frozen. RNA was extracted using Direct-zol™ RNA MiniPrep Plus (Zymo Research) and cleaned of potential DNA contamination with RNeasy MinElute Cleanup Kit (Qiagen). cDNA was synthesized with 10 ng of total RNA using the SMART-SeqTM v4 UltraTM Low input RNA Kit for Sequencing (Clontech Laboratories) and amplified by 7 cycles of locked-DNA PCR. The sequencing library was generated using the Nextera XT DNA Library Preparation (Illumina) according to the manufacturer's instructions. Libraries were submitted to quality control on a QIAxcel (Qiagen) and to quantification using a KAPA Library Quantification Kit (Kapa Biosystems). Libraries were subsequently normalized and pooled.