title: GSM9078881: OK6>mCherry-NLS,empty(attP2) type I MNs, bio rep 5; Drosophila melanogaster; RNA-Seq
library_name: GSM9078881
library_construction_protocol: Larvae were washed twice with PBS, followed by a wash in 70% ethanol and a final wash in PBS before being placed in a droplet of Schneider's complete media with 10% FBS for ventral nerve cord dissection. Ventral nerve cord tissue was sequentially recovered for each larva dissected in ice cold Schneider's media with 10% FBS, pooled by the appropriate genotype. Dissected tissue was rinsed three times in Schneider's media containing 1.5 mM EDTA and 1.5 mM L-Cysteine. Cells were dissociated in Schneider's media with 1.5 mM EDTA, 1.5 mM L-Cysteine, papain, and 1.25 mg/mL collagenase type-I at 25C on a nutator for 5 minutes and then triturated 30 times with a P200 pipette. The incubation and trituration step was repeated once before the tissue was sheared 7 times with a 27G1/2 needle. The dissociation reaction was quenched with the addition of Schneider's media with 10% FBS. Dissociated cells were passed through a 40 uM cell strainer, spun down at 0.7xg for 7 minutes before being resuspended in 1 mL of Schneider's media with 5% FBS. Cells were stained with 0.3 uM of Sytox Green to assess viability before being sorted on a BD Influx Cell Sorter or BD FACS Aria. Cells were sorted for high mCherry fluorescence and low Sytox Green fluorescence. Sorted cells were recovered in RLT buffer with 3.5 mM BME from the QIAGEN RNEasy Micro kit and processed according to manufacturer's instruction omitting the DNase digestion step. Libraries were prepared with the Takara SMART-Seq v4 Ultra Low Input RNA kit. cDNA was fragmented with a Covaris S220 Focused-ultrasonicator and libraries were constructed with the KAPA HyperPrep kit. Truncated universal stub adapters were ligated to the fragmented cDNA. PCR amplification with indexed primers was used to complete the adapter sequence and enrich the libraries for adapter-ligated cDNAs. Libraries were sequenced on a NovaSeq X instrument.