title: GSM1611554: Simulated Microgravity-2; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted from duplicates of cells subjected to stimulated microgravity and static control using RNA-STAT-60 (Tel-Test,Inc., Friendswood, TX) using the manufacturer’s instructions. Briefly, 1ml of RNA-STAT solution was added per 10^6 cells and homogenized for 5 minutes over ice. 1ml of chloroform was added, contents shaked vigorously and centrifuged at 12,000g at 4ºC for 15 minutes. The aqueous solution was transferred to a clean corex tube and 0.8 ml isopropanol added. After incubation of 10 minutes, the contents were centrifuged at 12,000g for another 10 minutes to precipitate the RNA. The RNA pellet was washed with 75% ethanol and centriguged ar 7500g for 5 minutes at 4ºC. The ethanol was aspirated and the RNA pellet dried. The RNA pellet was finally resuspended in DEPC water. RNA was converted into cDNA and  was end-repaired using repair solution (combination of T4 DNA polymerase, E. coli DNA Pol I large fragment and T4 polynucleotide kinase). A-tailing was obtained by incubating the end repaired DNA with dA-tailing mix. Blunt end ligation was performed by incubating the A-tailed DNA samples (1 μg) with adapters (IDT Inc., Coralville, IA), unmethylated version of Truseq adapters (Illumina Inc., San Diego, CA) for multiplexing. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the illumina hiseq2000 following the manufacturer's protocols.
attributes.tag: GEO Accession
attributes.value: GSM1611554