GSM8192366: astrocyte-24 h after injury-5; Drosophila melanogaster; RNA-Seq
Brains from adult flies (R86E01-Gal4/UAS-Redstinger) with no injury, 4 h after injury and 24 h after injury were dissected out in freshly prepared adult hemolymph saline (AHS) buffer, as previously described (Yang et al., 2016). Briefly, a total of ∼50 brains for each group was immediately transferred to an Eppendorf tube (1.5 mL) containing 200 µL AHS with Pronase (1 mg/mL; P5147, SigmaAldrich) and Dispase (1 mg/mL; LS02104, Worthington Biochemical Corporation). After tissue digestion for 30 min at 25°C, the enzyme medium was removed, followed by two brief, gentle washes with fresh AHS containing 2% PBS. Brain samples were then triturated by gently pipetting in 1 mL AHS through four fire-polished Pasteur pipettes with descending pore sizes (400 μm, 300 μm, 200 μm, and 100 μm) for a total of ∼20 min. Next, the dissociated cells were centrifuged for 10 min at 100 × g and the AHS was replaced with the same buffer to minimize any RNA content released from damaged cells during the trituration process. Finally, dissociated tissues were filtered through 45 μm filter and then put on ice. FACS sorting was performed according to the following gating procedure with 70 μm nozzle by BD FACS Aria: (1) an initial SSC-A/FSC-A gate to minimize debris; (2) a SSC-A/pe-Texa Ted-A gate to choose only red fluorescence positive cells. At this step, 1600 cells were sorted directly into ice-cold cell lysis buffer (oligo-dT30VN primer, ribonuclease inhibitor, dNTP mix) and stored at -80°C until all samples were ready for RNA extraction and cDNA amplification. Cell samples were sent to the Anoroad Company for RNA extraction. An Oligo-dT primer was introduced to the reverse transcription reaction for first-strand cDNA synthesis, followed by PCR amplification to enrich the cDNA and magbeads purification step was used to purify the products. Briefly, Smart-Seq2 method was used to amplify cDNA. Concentration and integrity of cDNA was assessed using Qubit 3.0 Flurometer (Life Technologies) and Agilent 2100 Bioanalyzer (Agilent Technologies) to ensure the cDNA length was around 1-2 kbp. After quality control, 40 ng cDNA was used to be fragmented at 350 bp by Bioruptor® Sonication System (Diagenode Inc.). To construct Illumina library, end repair, 3' ends A-tailing, adaptor ligation, PCR amplification and library validation were performed. PerkinElmer LabChip® GX Touch and Step OnePlus™ Real-Time PCR System were used for library quality inspection. Qualified libraries were loaded on Illumina Hiseq platform for PE150 sequencing.