GSM8001275: mouse NK rep3; Mus musculus; RNA-Seq
RNAs are amplified in situ, not extracted from the cells. 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. 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.