GSM5962693: J183_1; Mus musculus; RNA-Seq
Coverslips containing plated astrocytes were loaded with 10μg/ml Fluo-4 AM (#F14201, Thermo Fisher Scientific) mixed with 0.0001% pluronic acid (#P3000MP, Thermo Fisher Scientific) for 25min at room temperature then de-esterified for 10 mins with continual perfusion of external solution. Serum was removed 24 hours prior to experiments. External solution (Imaging saline, perfusion speed: 3ml/min) contained 140mM NaCl, 5.4mM KCl, 1mM MgCl2, 2mM CaCl2, 16mM glucose, 10mM HEPES, adjusted to pH 7.3. The calcium indicator-loaded coverslip was then transferred to an open bath chamber (RC-27, Warner Instruments) for imaging and sample collection. To minimize cell-to-cell fluorescence loading variation and animal and culture batch variations, the calcium signal in individual astrocytes was normalized against calcium signals induced by 100µM ATP at the end of each experiment. For single cell isolation, targeted single cells were identified based on real-time imaging results and collected with a pipet tip pulled from a glass microcapillary (#3450099, Thermo Fisher Scientific) controlled by a micromanipulator (Eppendorf, Transferman NK2). Immediately after isolating samples by gentle aspiration, samples were snap-frozen on an ethanol-dry ice mixture then briefly stored at -20oC prior to mRNA amplification Single cells were collected in 5x first strand buffer (ThermoFisher, supplied with #1808004) and snap frozen prior to storage. Single cells were then thawed and amplified using modifications to the antisense RNA (aRNA) amplification procedure (80). Alterations included using Beckman Coulter Ampure XP beads (#A63881) for all cleanups after second strand reaction or the Beckman Coulter Agencourt RNAClean XP (#A63987) following the in vitro transcription reaction. Cleaned double stranded cDNA or cleaned aRNA was eluted in small volumes to allow direct input into the next step without the need for an ethanol precipitation or speed vacuum concentration step. Amplification was restricted to two rounds to ameliorate the shortening effect of random primed based approaches as well as the severity of 3'bias of polyA selection amplification approaches. Second round aRNA was sheared based on overall size distribution assessed using an Agilent Tapestation 2200, with the shearing time adjusted for longer average aRNA products, prior to input into the Illumina TruSeq mRNA stranded library construction procedure (#20020594, set A indexes #20020492, and set B indexes #20020493). A total of 50ng of aRNA from a single sample was input into library construction. All other steps after shearing were followed according to the manufacturer's specifications. Following completion of library preparation, samples were multiplexed at 12 samples per lane and sequenced on a NextSeq 500 using the 150 cycle high output v2 kit (#FC-404-2002), allowing for paired end 2x75 base pair reads.