alias: E-MTAB-2672:WTCHG_47716_262
title: Illumina HiSeq 2500 paired end sequencing; Functional and genetic heterogeneity of distinctive leukaemic stem cell populations in CD34- human Acute Myeloid Leukaemia
design: Functional and genetic heterogeneity of distinctive leukaemic stem cell populations in CD34- human Acute Myeloid Leukaemia
library_construction_protocol: Mononuclear cells were isolated by Ficoll density gradient. In normal bone marrow samples, CD34+ cells were purified using CD34 Microbead Kit and MACS separation columns (Miltenyi Biotec, Bisley UK). Unseparated, CD34+ and CD34-deplete fractions were frozen in 90% FCS/10% DMSO and stored in liquid nitrogen, and subsequently thawed on the day of the experiment. Antibodies used for lineage-depletion cocktails and in flow cytometry panels in different experiments are shown in Supplemental Experimental Procedures. FACS analysis was carried out on either a Cyan ADP (Dakocytomation, Ely UK) or a BD LSR Fortessa (Becton Dickinson, Oxford UK). Flow sorts were carried out on a BD Aria III SORP (Becton Dickinson, Oxford UK). Total RNA was extracted from 5x103 - 4x104 sorted cells, quantified using the total RNA Pico or Nano Bioanalyzer chip (Agilent, Wokingham UK). All samples had a RIN score of ?7.0. 20pg to 4ng of total RNA was used for cDNA synthesis (SMARTer Ultra Low input RNA kit (Clontech, St Germain-en-Laye France). cDNA libraries were amplified and sequenced on the Illumina HiSeq 2000 and 2500 (paired-end, read length: 50 base pairs; Illumina, Saffron Walden, UK).
identifiers.namespace: PARESH VYAS GROUP, MOLECULAR HAEMATOLOGY UNIT, THE WEATHERALL INSTITUTE OF MOLECULAR MEDICINE
identifiers.id: E-MTAB-2672:WTCHG_47716_262
identifiers.namespace: Paresh Vyas group, Molecular haematology Unit, The Weatherall Institute of Molecular Medicine
identifiers.id: E-MTAB-2672:WTCHG_47716_262