title: GSM2113743: LIGR-Seq_AMT_Ligase_Rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Immediately after incubation, reactions were placed on ice and RNA was phenol-chloroform extracted. After washing in 70% ethanol, the RNA was resuspended in 20 μL of TS buffer. For crosslink reversal, RNA was resuspended in TS buffer in open eppendorf tubes were placed on ice. The RNA was irradiated for 5 min intervals for 10 min at 254 nm at a distance of ~ 15 cm from the UV bulb. AMT was removed from samples by ethanol precipitation and the RNA was resuspended in 10 μL of water. The SMARTER strand library preparation kit (Clontech), which is well suited for low amounts of input, was used to prepare libraries from RNA samples according to the manufacturer’s instructions with a few modifications. Briefly, first strand cDNA synthesis was performed using a primer that adds a universal sequence to the 5´ end of the first strand, and using a reverse transcriptase that adds several nucleotides to the end of the cDNA. A universal forward primer was annealed to the added nucleotides for synthesis of the second cDNA strand. Primers specific to added sequences from the first and second strand cDNA synthesis were used for library amplification, where the reverse primer includes a barcode index allowing multiplexing during sequencing. Libraries were amplified for 20 cycles for replicate 1 and 17 cycles for replicate 2. For extraction and clean-up of nucleic acid from the RT reaction and PCR reaction with Solid Phase Reversible Immobilization (SPRI) AMPure beads, a 1:2 ratio of sample:beads was used to obtain fragments < 100 bp (the ratio of sample to beads preferentially selects an empirically determined size range of nucleic acid). Following library preparation, samples were separated on a 6% TBE 1.0 PAGE gel (Novex) at 150 V, 16 A. Products above 200 nt were excised and eluted overnight in 30 μL of H20 at 10oC. Size distribution was verified using a Bioanalyzer.
attributes.tag: GEO Accession
attributes.value: GSM2113743