GSM7835882: S2 cells, RNA-seq, replicate 1; Drosophila melanogaster; RNA-Seq
For each cell line RNA was made from 4 separate tissue culture flasks to generate four independent replicates. For each RNA prep 1Î107 cells spun down (1000 g, 3 min), washed twice with ice-cold PBS, and resuspended in 1 mL of ice-cold PBS, transferred to a 1.5 mL Eppendorf tube and spun down at full speed in a microfuge for 2 min at 4 ℃. The pellet was resuspended in 100 µL PBS. Added 1 mL of Trizol to the cell suspension and vortexed for 1 min. Incubated the samples at RT for 15 min. Added 200 µL of chloroform and vortexed for 2 min. Centrifuged the samples at 13,200 rpm 15 min at 4 ℃. Carefully transferred the top aqueous layer containing the RNA to a fresh tube. Measured the RNA concentration using the broad range Qubit RNA kit and a Qubit 3.0 fluorimeter (Invitrogen). An aliquot of the total RNA was further purified using the Qiagen RNeasy Micro kit following the manufacturer's directions. Libraries were made using the Illumina TruSeq Stranded mRNA sample prep kit, and then run on a NovaSeq 6000 using a SP 200 kit by the NICHD Molecular Genomics Core.