GSM3756480: A_CD34+38-_CB16a; Homo sapiens; RNA-Seq
RNA was extracted from snap-frozen cells using the All prep DNA/RNA Mini Kit (Qiagen, 80204 - lysis steps) and  mirVana miRNA Isolation Kit (Life Technologies, AM1560 - extraction steps) followed by ethanol precipitation. RNA was quantified using an Agilent Bioanalyzer (Life Technologies) and 500 ng of total RNA was ribosomal RNA (rRNA) depleted using a NEBNext rRNA Depletion Kit (New England BioLabs, E6310L). First strand cDNA was generated using a Maxima H minus First Strand cDNA Synthesis Kit (Thermo Scientific, K1652) with added Actinomycin D (1 μg, Sigma, A9415). The product was puri- fied using in-house-prepared 20% PEG in 1 M NaCl Sera-Mag bead solution at a 1.8× ratio and then eluted in 35 μl of Qiagen EB buffer. Second-strand cDNA was synthesized in a 50 μl volume using SuperScript Choice System for cDNA Synthesis (Life Technologies, 18090-019) with 12.5 mM GeneAmp dNTP Blend with dUTP. Double-stranded cDNA was purified with 20% PEG in 1 M NaCl Sera-Mag bead solution at a 1.8× ratio and eluted in 40 μl of Qiagen EB buffer, and fragmented using Covaris E220 (55 s, 20% duty factor, 200 cycles per burst). Sheared cDNA was end repaired/phosphorylated, single A-tailed, and adaptor ligated using custom reagent formulations (New England BioLabs, E6000B-10) and in-house-prepared Illumina forked adaptor. PEG (20%) in 1 M NaCl Sera-Mag bead solution was used to purify the template between each of the enzymatic steps. To complete the pro- cess of generating strand directionality, adaptor-ligated template was digested with 5 U of AmpErase Uracil N-Glycosylase (Life Technologies, N8080096). Libraries were then indexed and PCR amplified using Phusion Hot Start II High Fidelity Polymerase (Thermo Scientific, F 549-L).
GEO Accession
GSM3756480