title: GSM1289098: sRNAs from KSHV-infected BC-3 cells; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted from cells using Tri-reagent (MRC, Inc) Libraries were prepared as previously described with some modifications (Pfeffer, S. 2007. Identification of virally encoded microRNAs. Methods Enzymol. 427:51-63). Briefly, 10 μg of total RNA from BCBL-1, BC-3 and DG-75-K10/12 were size fractionated on a 17.5% urea denaturing polyacrylamide gel. The 19-33 nt band was excised, crushed and solved in 2 volumes of 0.3 M NaCl by overnight agitation at 4°C. Small RNAs were sequentially ligated to degenerate 3' and 5' adapters, with T4 RNA Ligase 2 and T4 RNA Ligase 1, respectively, to minimize ligation bias due to secondary structure formation. Prior to this, the 3' adapter was adenylated using the 5' DNA adenlylation kit according to the manufacturer's recommendations (New England Biolab, #E2610S). After each ligation step, the product was purified in denaturing polyacrylamide gel. In order to generate cDNA libraries, RNA was reverse transcribed with SuperScript III RT (Invitrogen) and primer GCCTGGCACCCGAGAATTCCA, then amplified by PCR with forward primer AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA and different reverse primers for multiplex sequencing (RPI1, RPI4 and RPI5, Illumina PCR primers) during 14 or 17 cycles (for BC-3, or DG-75-K10/12 and BCBL-1 libraries respectively) (45 sec at 94°C, 85 sec at 50°C and 60 sec at 72°C). PCR products were sent for large-scale sequencing
attributes.tag: GEO Accession
attributes.value: GSM1289098