GSM5583722: ImmunoMPRA E15.5 Pax6+ rep1; Mus musculus; OTHER
The somatosensory cortex of in utero electroporated E15.5 mouse embryos was isolated and the electroporated area dissected. Single cell suspension was prepared using the papain-based neural dissociation kit (Miltenyi Biotec, Cat. N: 130-092-628) according to the manufacturer protocol with reduced dissociation times (5 min. instead of 15 min. at 37˚C). Cell number as well as the viability was assessed using the Countess™ II Automated Cell Counter (Invitrogen). Dissociated cells were and immediately FAC-sorted at a BD FACSAria Fusion (BD Bioscience) with four lasers (405, 488, 561, 640) using a 100µm nozzle. Fixed sorted cells were digested with Proteinase K (Proteinase K (New England Biolabs, Cat. N: P8107S) at 55˚C for 1 h and decrosslinked at 65 ˚C for 15 min. RNA and DNA were extracted using the Quick-DNA/RNA Microprep Plus Kit (Zymo Research; Cat. N: D7005). Purified RNA was treated with TURBO DNase (Thermo Fisher; Cat. N: AM1907) and reverse transcribed with Maxima H Minus RT (Thermo Fisher; Cat. N: EP0753) using Oligo(dT)18 Primer (Thermo Fisher; Cat. N: SO131). cDNA was purified with 1.5X vol. AMPure XP magnetic beads (Agencourt, Cat. N: A63881) and stored at -20 °C for later use. Libraries from DNA and cDNA were prepared in the same way. Briefly, UMIs were added (98°C 30s; {98°C 10s, 65°C 30s, 72°C 1min} x3; 72°C 3min; Hold at 4°C) using the primers RV_univ_MPRA (CGACGCTCTTCCGATCT) and FWD_mScar_Tn7_10UMI_3 (0.5 µM each; GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG NNNNNNNNNN TCGACCGCAAGTTGGAC). P7 and P5 sequencing adapters were attached in two separate PCRs (98°C 30s; {98°C 10s, 65°C 90s} x(10+X); 72°C 5min; Hold at 4°C) using indexed Ad2.X(CAAGCAGAAGACGGCATACGAGAT XXXXXXXX GTCTCGTGGGCTCGGAGATGT) and P5NEXTPT5 primers (0.1 µM each; AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCG*A*T*C*T). Required PCR cycle numbers were estimated by qPCR and using 1/10 of the second PCR product as input. All PCRs were performed in 1X NEBNext® Ultra™ II Q5® Master Mix (New England Biolabs; Cat. N: M0544), and PCR products were purified with 0.8X - 1.2X vol. of AMPure XP magnetic beads.