GSM3374619: lna_cell_jurkat_tcr_resampled; Homo sapiens; RNA-Seq
10x Illumina libraries at 2nM concentration were diluted 20 fold and PCR amplified with Truseq Pcr F and R oligo for 14 cycles with 1 unit of Phusion.  PCR results were confirmed by gel electrophoresis and the remaining sample was Ampure XP purified at a ratio of 1.8x bead/pcr volume.  Samples were eluted in 10µl and molarity was determined using a Qubit.  10µl of 100nM library was mixed with 1 µl of 10µM LNA molecule in anneallng buffer (10mM Tris 8.0, 50mM NaCl) and heated to 98˚ for 10 minutes followed by slow cooling to 59˚-64˚ depending on the LNA and held O/N at the annealing temperature.  Annealed DNA was added in equal volume to washed  Dynabead M270 streptavidin beads at the annealing temperature.  Beads were incubated for 15 minutes with gentle shaking.  Beads were washed 5x in bind and wash buffer at the annealing temperature.  Beads were eluted in 20 µl of elution solution (50mM NaCl, .1mM EDTA) heated for 10 minutes at 100˚ and centrifuged briefly to pellet the streptavidin-bound LNA molecules which inhibit PCR, this elution was done 2 times to ensure all the DNA was removed from the LNA.   10µl of streptavidin purified input DNA was added to PCR using Truseq Pcr F and R for 8 cycles.  PCR products were ampure purified, confirmed on the Tapestation D1000, and quantified by the Qubit and submitted for sequencing. Read 1 positions 1-16 contain the 16bp cell barcode, and positions 17-26 contain a 10bp UMI. The remainder of read 1 contains the 5' of the TCR alpha or beta cDNA. Read 2 also contains the cDNA.
GEO Accession
GSM3374619