title: GSM8680431: LPS; Mus musculus; RNA-Seq
library_construction_protocol: Cells were processed for RNAseq in FACS buffer (2% fetal bovine serum + 0.1% NaN3 in phosphate buffered saline (PBS); all reagents purchased from Sigma unless otherwise specified). Single cell suspensions generated from whole lungs were quantified and viability tested using a LUNA-FLTM Dual Fluorescence Cell Counter (Logos Biosystems, Annandale, VA). Single cells were isolated using a 10x Chromium controller per manufacturer's suggested protocol (10x Genomics, Pleasanton, CA). Following cell capture, the gel beads in emulsion/sample solution were recovered and placed into strip tubes. Reverse transcription was performed on a thermocycler (C100 TouchTM Thermal cycler, Bio-Rad, Hercules, CA) per recommended protocol followed by cDNA amplification. Amplified products were solid phase reversible immobilization (SPIR) bead-purified and evaluated by Fragment Analyzer (Agilent, Santa Clara, CA). Twenty-five percent of the cDNA volume was subjected to fragmentation and double-sided SPRIselect (Beckman /Coulter, Indianapolis, IN) was used for PCR purification. PCR products were purified, quantified, and library size distribution determined by fragment Analyzer. Libraries were sequenced per the manufacturer's suggested parameters on a NextSeq500 sequences to an average depth of 50,000 reads per cell.