GSM4144497: Cell56_Skininnervating; Mus musculus; RNA-Seq
Single-cell libraries were generated according to the Smart-Seq2 protocol (Picelli et al. Nature Methods 2014 and Trombetta et al. Current Protocols in Molecular Biology 2014). Each fluorescent cell was pipetted up and down the micropipette three times to remove unwanted contamination before being ejected into 10 μl of cold RLT Buffer (Qiagen) supplemented with 1% β-mercaptoethanol in a 96-well plate, and snap-frozen on dry ice and stored at -80ºC. RNA from single-cell lysates was purified using AMPure RNA Clean Spri beads (Beckman Coulter) at a 2.2x volume ratio. Following AMPure Bead cleanup, RNA was eluted in water and mixed with oligo-dT primer (SmartSeq2 3' Oligo-dT Primer), dNTPs, and RNase inhibitor at 72­ºC for 3 minutes on a thermal cycler to anneal the 3' primer to polyadenylated mRNA. Reverse transcription was carried out in a master mix of Maxima RNaseH-minus RT enzyme and buffer, MgCl2, Betaine, RNase inhibitor, and a 5' template switch oligonucleotide using the following protocol: 42ºC for 90 minutes, followed by 10 cycles of 50ºC for 2 minutes, 42ºC for 2 minutes, and followed by inactivation at 70ºC for 15 minutes. Whole transcriptome amplification was achieved by addition of KAPA HiFi HotStart ReadyMix and IS PCR primerto the reverse transcription product and amplification on a thermal cycler using the following protocol: 98ºC for 3 minutes, followed by 21 cycles of 98ºC for 15 seconds, 67ºC for 20 seconds, 72ºC for 6 minutes, followed by a final 5-minute extension at 72ºC. Libraries were purified using AMPure XP SPRI beads at a volume ratio of 0.8x followed by 0.9x. Library size was assessed using a High-Sensitivity DNA chip (Agilent Bioanalyzer), confirming the expected size distribution of ~1000-2000 bp. Tagmentation reactions were carried out with the Nextera XT DNA Sample Preparation Kit (Illumina) using 250 pg of cDNA per single cell as input, with modified manufacturer's instructions as described. Libraries were purified twice with AMPure XP SPRI beads at a volume ratio of 0.9x, size distribution assessed using a High Sensitivity DNA chip (Agilent Bioanalyzer) and Qubit High-Sensitivity DNA kit. Libraries were pooled and sequenced using NextSeq500/550 High Output v2 kits (75 cycles, Illumina) using 30-30 paired end sequencing with 8-mer dual indexing
GEO Accession
GSM4144497