GSM9293323: Stellate Ganglion, plate 9; Mus musculus; RNA-Seq
The plates were centrifuged at 200g for 2 min and snap frozen on dry ice. Each well received 7.5 μl of lysis buffer [0.2% Triton X-100 (Sigma), SUPERaseIN (1 U/μl) (Thermo Fisher Scientific), 2 mM deoxyribonucleotides (dNTPs) (Thermo Fisher Scientific), and 2 μM reverse transcriptase (RT) primer (Integrated DNA Technologies)]. Primer annealing was performed at 72°C for 3 min. Reverse transcription was performed by adding 7.5 μl of RT mix to each well [2 M betaine (Affymetrix), 2× Protoscript Buffer (New England Biolabs), 12 mM MgCl2 (Thermo Fisher Scientific), 10 mM dithiothreitol (Thermo Fisher Scientific), 5.3 U of Protoscript II Reverse Transcriptase (New England Biolabs), 0.53 U of SUPERaseIN (Thermo Fisher Scientific), and 2 μM Template Switching Oligo (Integrated DNA Technologies; table S6)]. Reverse transcription was performed at 42°C for 90 min, followed by 10 cycles of 50°C for 2 min, 42°C for 2 min, 70°C for 10 min, followed by a 4°C hold. Excess primers were removed by adding 2 μl of Exonuclease I (Thermo Fisher Scientific) mix to each well (1.875 U of ExoI in water) and incubating at 37°C for 30 min, 85°C for 15 min, 75°C for 30 s, and 4°C hold. The cDNA was pooled for PCR and library construction. Pooled, 3'-end sequencing libraries were sequenced on an Illumina NextSeq 500/550 platform.