GSM8005129: WGA-Pd rep2; Homo sapiens; RNA-Seq
RNA was amplified in-situ, not extracted from the cultured cells. For AMOUR: Cells (> 97% viability) were fixed with 4% PFA and immobilized with Concanavalin A beads. The bead-bound cells underwent three washes with AMOUR-wash buffer (PBS, 1 mM ATP, 0.2 U μL-1 RNase inhibitor). Subsequently, the beads were resuspended in PBS with RNase Inhibitor (40 U μL-1) and annealed with T7N9 oligo (0.25 μM). The reaction mixture was incubated at 42 °C for 2 min, followed by ramping to 25 °C and a 5-min hold. After washing, linear amplification with T7 RNA Polymerase occurred at 37 °C for 2 h with shaking at 1,200 rpm. The resulting RNA was purified using Dynabeads MyOne Silane beads. For GWA-Pd and WGA-T: HEK293T cells were collected with (WGA-T) or without 0.25% trypsin treatment (WGA-Pd), and blocked with Egg white avidin and yeast tRNA. Following buffer removal, 500 µg of biotin-WGA was added to the cells and incubated at 37 ℃ for 15 min. After centrifugation at 500 × g, 4 ℃ for 5 min to eliminate excess biotin-WGA, cells were washed with PBS. Lysis was performed with ice-cold lysis buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 1% DDM, and 0.001% GDN with 0.1% CHS, 200 U mL-1 RNase inhibitor, 1 × proteinase inhibitor). Dynabeads MyOne Streptavidin C1 were utilized for capturing membrane-associated RNA, extracted using Buffer RLT, and subsequently purified using silane beads. For RT-Amp: Cells were harvested, fixed, and immobilized utilizing ConA beads, followed by T7N9 oligo annealing through the "AMOUR" strategy. Reverse transcription (RT) was performed on beads employing HiScript II Reverse Transcriptase. Following RT, the cDNA product underwent purification with the DNA Clean Concentrator. Subsequent second-strand synthesis was achieved with Klenow exo- and P5N6 Oligo, and dsDNA was purified using silane beads. Linear amplification was executed using T7 RNA Polymerase. The resulting amplified RNA was purified with silane beads. The purified RNA was ligated to 3'adapter and reverse transcribed. The cDNA was ligated with cDNA adapter and subjected to limited cycle PCR, ready for sequencing.