GSM5532212: Matrigel3; Homo sapiens; RNA-Seq
Samples were resuspended in 5 µl 1X NEBNext Cell Lysis Buffer of the NEBNext Single Cell/Low Input RNA Library Prep Kit (NEB, E6420). After 5 min incubation at RT, 2.5X RNA XP Clean Up Beads (Beckman Coulter, A63987) were added to the sample by pipetting up and down. Then, samples were incubated for 5 min at RT, the beads were pelleted on a magnet, the supernatant was removed, the samples were washed twice with 80% ethanol while remaining on the magnet, and finally the beads were resuspended in 8 µl nuclease-free water. The reverse transcription was conducted according to the manual of the NEBNext Single Cell/Low Input RNA Library Prep Kit in the presence of the beads The lysates were thawed on ice and the RNA precipitated by adding 0.1 volume 3 M sodium acetate (pH 5.5) and 2.5 volumes 100% cold ethanol. After Incubation at − 80 °C for 30 min, the RNA was recovered by centrifugation at 16,000×g for 30 min at 4 °C. The RNA pellet was washed with 1 mL 80% cold ethanol, centrifuged at 16,000×g for 5 min at 4 °C, the ethanol solution carefully removed by aspiration and the RNA pellet dried. The pellet was resuspended in 5 µl 1X NEBNext Cell Lysis Buffer of the NEBNext Single Cell/Low Input RNA Library Prep Kit (NEB, E6420). After 5 min incubation at RT, 2.5X RNA XP Clean Up Beads (Beckman Coulter, A63987) were added to the sample by pipetting up and down. Then, samples were incubated 5 min at RT, the beads were pelleted on a magnet, the supernatant was removed, the samples were washed twice with 80% ethanol while remaining on the magnet, and finally the beads were resuspended in 8 µl nuclease-free water. The reverse transcription was conducted as described in the manual of the NEBNext Single Cell/Low Input RNA Library Prep Kit in the presence of the beads.