GSM5952508: CEPT-E6D7_8; Homo sapiens; RNA-Seq
oligo-dt primers; Samples were harvested and lysed in TCL lysis buffer (Qiagen). Lysates were transferred to TurboCapture 384 format plates (Qiagen) and incubated at RT 1 hr at 100 rpm for polyA RNA capture. Cell lysate then washed with a binding buffer (20 mM Tris-HCl, pH 7.5 at 25 ⁰C, 500 mM NaCl, 1 mM EDTA, 0.1% SDS) twice and 20 µL probes mixture was added into each well with 0.05 pmol/oligo in binding buffer. The binding mixture was incubated at 65 ⁰C for 10 minutes and gradually the temperature was lowered to 45 ⁰C by every 3 degree/per minute rate before 1 hr incubation at 45 ⁰C in the thermocycler. The ligation step was performed after three times wash with wash buffer (20 mM Tris-HCl, pH7.5 at 25⁰C, 100 mM NaCl, 1 mM EDTA, 0.1% Tween 80) and one time wash with ligation buffer (40 mM Tris-HCl, pH7.5 at 25⁰C, 10 mM MgCl2, 10 mM DTT, 10 µM ATP). A 20 µL ligation mixture containing 5U (Weiss unit) of T4 DNA ligase in ligation buffer was added into each well. After ligation at 37 ⁰C for 1 hr, the wells were washed three times with wash buffer. The final products were eluted with 25 µl elution buffer. Final products of 5 µL/sample were used for PCR amplification with 0.05 µM barcoded primers and AmpliTaqGold DNA polymerase (0.06 U/reaction, Thermo Fisher Scientific) in buffer I supplied by the manufacturer. The dual indexed PCR products were then pooled as one single sample and purified using size-selected column to remove primer dimers (Zymo research). The purified samples were quantified and sequenced on NextSeq 550 using custom sequencing primers.