Illumina HiSeq 2000 paired end sequencing; RNA-Seq analysis of LT-HSC isolated from fetal liver and adult bone marrow.
RNA-Seq analysis of LT-HSC isolated from fetal liver and adult bone marrow.
Total adult bone marrow (ABM) cells were flushed from femurs and tibiae of male C57BL/6J mice, pooled, washed twice with phosphatebuffered saline (PBS; Gibco Invitrogen, CA) containing 0.1% bovine serum albumin (BSA; Sigma). Lineage negative cell isolation was performed using lineage cell depletion kit (Miltenyi Biotec, Germany). To isolate LT-HSCs from ABM, resulting Lin cells were stained with FITC conjugated antiSca1, PE conjugated antickit, APC conjugated antilineage antibody cocktail (BD Pharmingen, San Diego, CA), PerCP/Cy5.5 conjugated anti-CD150 and APC conjugated anti-CD48 antibodies. Cells were incubated on ice for 30 minutes. FL tissues were obtained from C57BL/6J embryos dissected at embryonic day (E) 14.5 (14 days after vaginal plug was observed). Ter-119 positive erythrocytes and erythrocyte progenitors were depleted using MACS columns (Miltenyi Biotec, Germany) and stained with Alexa Fluor 488 conjugated anti-lineage antibody cocktail (containing CD4, CD5, CD8a, CD45R, Ter-119, GR-1 antibodies), PE conjugated anti-CD11b, APC conjugated anti-Sca-1, PE-Cy7 conjugated anti-CD150 and Alexa Fluor 488 conjugated anti-CD48. Cells were incubated on ice for 30 minutes. After incubation of cells with antibodies, cells were washed once with PBS. HSCs were sorted by fluorescence-activated cell sorting (FACS), using a FACS ARIAIII (Becton Dickinson). HSCs were directly sorted in QIAzol lysis buffer from miRNeasy Micro Kit (QIAGEN) and stored at -80c until isolation. Fraction of cells were used to checked the purity which was >95 percent. Total RNA isolation was isolated from the indicated populations using miRNeasy Micro Kit (QIAGEN) according to the manufacturer's protocol. DNase treatment was performed using RNAse-free DNase Set (Qiagen). Total RNA from FL and BM HSC was amplified using Ovation RNA-seq V2 system (NuGen technologies, CA) as per manufacturers instructions. Amplified cDNA (1ug) was sheared using the Covaris system (Covaris, MA) and fragments of 200bp (180-220bp) selected to construct a cDNA library. The fragmented DNA is combined with End Repair Mix, incubate at 20C for 30 min. Purify the end-repaired DNA with QIAquick PCR Purification Kit(Qiagen),then add A-Tailing Mix, incubate at 37C for 30 min. Combine the purified Adenylate 3'Ends DNA, Adapter and Ligation Mix, incubate the ligation reaction at 20C for 15 min. Adapter-ligated DNA is selected by running a 2% agarose gel to recover the target fragments. Purify the gel with QIAquick Gel Extraction kit (QIAGEN). Several rounds of PCR amplification with PCR Primer Cocktail and PCR Master Mix are performed to enrich the Adapter-ligated DNA fragments. Then the PCR products are purified with Ampure XP Beads (AGENCOURT). The Qualified libraries will amplify on cBot to generate the cluster on the flowcell (TruSeq PE Cluster Kit V3cBotHS,Illumina). And the amplified flowcell will be subjected to 2*90 bp paired-end sequencing on the HiSeq 2000 System (TruSeq SBS KIT-HS V3,Illumina).
Experimental Factor: fetus
organism part