GSM3039093: sample101: dark 30m, corn oil; Mus musculus; RNA-Seq
RNA was extracted using the Qiagen Rneasy kit with the DNase step according to instructions of the manufacturer. Libraries were constructed using the Illuminia LT or HT TruSeq Stranded Total RNA kit according to the instructions of the manufacturer except that all volumes were reduced to 1/3 of their recommended amount. For captured libraries, 250ng of pooled libraries were heated to 95C to denature DNA and then incubated with 250ng ARG-seq or eRNA-seq RNA baits overnight at 65C in hybridization buffer (2.5ug Cot1 DNA (ThermoFisher), 2.5ug Salmon Sperm DNA (ThermoFisher), 15mM p5 blocking primers, 15mM p7 blocking primers, 5X SSPE (ThermoFisher), 5X Denhardt's Solution (ThermoFisher), 0.133% SDS). Blocking primers are: p5-AATGATACGGCGACCACCGAGATCTACAC, ACACTCTTTCCCTACACGACGCTCTTCCGATC/3InvdT/ p7-CAAGCAGAAGACGGCATACGAGAT, GTGACTGGAGTTCAGACGTGT GCTCTTCCGATC/3InvdT/ Primers for amplification are: p5-AATGATACGGCGACCACCGAGA, p7-CAAGCAGAAGACGGCATACGAG. Hybridized samples were incubated with MyOne Streptavadin T1 Dynabeads (Invitrogen) in binding buffer (1M NaCl, 10mM Tris-HCl pH 7.5, 1mM EDTA). Beads were washed once in 1x SCC, 0.1% SDS at room temperature and three times in 0.1x SCC 0.1% SDS at 65C. Captured libraries were eluted with 0.1M NaOH and neutralized with 1M Tris-HCl pH 7.5. Libraries were then purified using the Qiagen MinElute PCR cleanup kit and re-amplified using Herculase II Fusion polymerase (Agilent).
GEO Accession
GSM3039093