GSM2631409: H5N1_3hr [miRNA]; Homo sapiens; miRNA-Seq
The macrophages were infected with H1N1 and H5N1 separately at a MOI of two. The cells were washed with warm culture medium and incubated in macrophage serum free medium (Invitrogen) supplemented with 0.6 mg/l penicillin and 60 mg/l streptomycin. The total RNA was extracted from cells at 1-, 3-, and 6-hr post-infection using the mirVana miRNA Isolation Kit (Ambion, Inc. TX, USA). Small RNA library was prepared from rRNA-depleted RNA from 1 µg of total RNA. The 3’ adapter (5’-rAppAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG/3ddC/-3’, 3ddC represents a 3’ OH blocking group) that had an adenylated 5’-end and a ddC 3’-end was ligated to the 3’ end of the RNA using T4 RNA Ligase 2 (NEB, Ipswich, MA). The 5’ adapter (5’-rArCrArCrUrCrUrUrUrCrCrCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrU-3’) ligation was performed using T4 RNA Ligase 1 (NEB, Ipswich, MA). The ligation product was used as template for cDNA synthesis using SuperScript II Reverse Transcriptase (Invitrogen) and an oligonucleotide with sequence complementary to 3’ adapter as RT primer (5’-CTCGGCATTCCTGCTGAACCGCTC–3’).
GEO
GSM2631409