title: GSM1620172: Unstimulated Mouse Bone Marrow Derived Dendritic Cells (t0); Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted with QIAzol reagent following the miRNeasy kit’s procedure (Qiagen), and sample quality was tested on a 2100 Bioanalyzer (Agilent). We prepared the RNA-A+-Seq libraries using the ‘dUTP second strand (strand specific) protocol as described in (Levine et al 2010). Briefly, extracts were treated with DNase (Ambion 2238). Polyadenylated RNAs were selected using Ambion's MicroPoly(A)Purist kit (AM1919M) and RNA integrity confirmed using Bioanalyzer (Agilent). RNA was fragmented by incubation in RNA fragmentation buffer (Affymetrix) at 80 degrees for 4 minutes. Fragmented RNA was mixed with 3 μg random hexamers (Invitrogen), incubated at 70 °C for 10 min, and placed on ice briefly before starting cDNA synthesis. We synthesized first-strand cDNA with this RNA primer mix by adding 4 μl 5× first-strand buffer, 2 μl 100 mM DTT, 1 μl 10 mM dNTPs, 4 μg of actinomycin D, 200 U SuperScript III and 20 U SUPERase-In, incubating at room temperature for 10 min followed by 1 h at 55 °C. We synthesized second-strand cDNA by adding 4 μl of 5× first-strand buffer, 2 μl of 100 mM DTT, 4 μl of 10 mM dNTPs with dTTP replaced by dUTP (Sigma), 30 μl of 5× second-strand buffer, 40 U of Escherichia coli DNA polymerase, 10 U of E. coli DNA ligase and 2 U of E. coli RNase H, and incubating at 16 °C for 2 h. cDNA was eluted using the Qiagen MiniElute kit with 30 μl of the manufacturer's EB buffer. DNA ends were repaired using dNTPs and T4 polymerase (NEB), followed by purification using the MiniElute kit. Adenine was added to the 3′ end of the DNA fragments using dATP and Klenow exonuclease (NEB; M0212S) to allow adaptor ligation, and fragments were purified using MiniElute. Adaptors were ligated and incubated for 15 min at room temperature (25 °C). Phenol/chloroform/isoamyl alcohol (Invitrogen 15593-031); extraction followed to remove the DNA ligase. The pellet was then resuspended in 10 μl EB buffer. The sample was run on a 3% agarose gel (Nusieve 3:1 agarose, Lonza) and a 160–380 base pair fragment was cut out and extracted. PCR was performed with Phusion High-Fidelity DNA Polymerase with the manufacturer's GC buffer (New England Biolabs) and 2 M betaine (Sigma). PCR conditions were 30 s at 98 °C; 16 cycles of 10 s at 98 °C, 30 s at 65 °C, 30 s at 72 °C; 5 min at 72 °C; forever at 4 °C. Products were run on a polyacrylamide gel for 60 min at 120 V. The PCR products were cleaned up with Agencourt AMPure XP magnetic beads (A63880) to completely remove primers and the product was submitted for Illumina sequencing.
attributes.tag: GEO Accession
attributes.value: GSM1620172