GSM2756758: indrops_Mecp2WT_rep7; Mus musculus; RNA-Seq
SUN1-GFP expressing nuclei were isolated as previously described (Mo et al., 2015) using GFP antibodies (Fisher G10362) coupled with Protein G Dynabeads (Invitrogen 10003D). RNasein Plus RNase Inhibitors (Promega N2611) were added to the buffers to prevent degradation of RNA. Co-encapsulation into microfluidic droplets of nuclei, polyacrylamide gels containing barcoded reverse transcription primers, reverse-transcriptase enzyme, and lysis buffer were performed as previously described to generate barcoded cDNA from single nuclei (Klein et al., 2015; Zilionis et al., 2017). We added 57µL of FD pre-mix (5µL 10X FD buffer; 45µL ddH20; 3µL ExoI (NEB M0293S, 20U/µL); 4µL HinFI (Thermo FD0804) to every 40 µl of sample. Samples were filtered through a 0.45 µm cellulose acetate filter, and incubated at 30 min at 37 °C. Samples were purified with 1.2X volume Agencourt AMPure XP beads (Beckman Coulter A63881). Second strand complementary DNA synthesis was performed by adding 10X NEB Second Strand Synthesis Buffer and NEB Second Strand Synthesis Enzyme Mix (New England Biolabs E6111S) and incubated at 16°C for 2.5 hours, then heat inactivated at 65 °C for 20min. In vitro transcription was performed with NEB T7 High Yield 10X Buffer, dNTPs, NEB T7 High Yield Enzyme Mix (NEB E2040S) and incubating at 37 °C for 13 hours. Samples were purified with 1.3X volume AMPure beads. RNA was fragmented with Fragmentation reagent (Ambion AM8740) by incubating at at 70°C for 3 min, stopped with the STOP mix, and purified with the 1.2X volume AMPure beads. Reverse transcription was performed using random hexamers: Fragmented RNA were incubated with 10 µM primers (TCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNN) and 10mM dNTPs for 3 min at 70°C. Then added RNase Inhibitor, 5X PrimeScript Buffer, 0.5 µL PrimeScript Reverse Transcriptase (Takara Clontech 2680A), and incubated at 30 °C for 10 min, 42 °C for 1 hour, then 70 °C for 15 min. The product was purified with 1.2X volume AMPure beads. The product was amplified using 2X KAPA HiFi HotStart ReadyMix (KAPA Biosystems KK2601), 5 µM primer mix (PE1: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGA; PE2: CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAAC) using the following conditions 98°C 2 min, 2 cycles of (98°C 20sec, 55°C 30sec, 72°C 40sec), 9 to 14 cycles of (98°C 20sec, 65°C 30sec, 72°C 40sec), 72°C 5 min, 4°C hold.
GEO Accession
GSM2756758