title: GSM4433466: Cop1 KO rep2; Mus musculus; RNA-Seq
library_construction_protocol: Microglia were isolated from Copwt/wt Cx3Cr1-CreERT2-eYFP and Copfl/fl Cx3Cr1-CreERT2-eYFP mouse forebrains and quality controlled as previously described (Srinivasan et al., 2016). All steps were carried out at 4°C. Briefly, after perfusion with ice-cold PBS, cortex and hippocampi were dissected and triturated at single cell suspension. After obtaining the cell pellet post Percoll gradient step, cells were washed and resuspended in 4 ml Hibernate A-LF (BrainBits) and stained with Calcein Blue, AM (C1429, ThermoFisher), which served as a viability probe. Filtered cells were then FACS sorted, using eYFP as a microglia marker in addition to Calcein Blue as a viability probe. Animals were always processed in pairs (1 Copwt/wt Cx3Cr1-CreERT2-eYFP and 1 Copfl/fl Cx3Cr1-CreERT2-eYFP) and sorted simultaneously in two BD FACS ARIA sorters. Microglia (Calcein Blue+, eYFP+) were collected directly in 350 μl RNA lysis buffer (RLT buffer from the Qiagen RNeasy Mini kit) and processed for RNA extraction Sample processing for single-cell RNA-sequencing was done using Chromium Single Cell 3' Library and Gel bead kit v2 (PN-120237) following manufacturer's user guide (CG00052, 10x Genomics, Pleasanton, CA). The cell density and viability of the single-cell suspension were determined by Vi-CELL XR cell counter (Beckman Coulter, Pasadena, CA). All of the samples had >80% viable cells. The total cell density was used to impute the volume of single cell suspension needed in the reverse transcription (RT) master mix, aiming to achieve ~ 6,000 cells per sample. cDNAs and libraries were prepared following manufacturer's user guide (10x Genomics). cDNA amplification and indexed libraries were prepared using 12 and 14 cycles of PCR, respectively. Libraries were profiled by Bioanalyzer High Sensitivity DNA kit (Agilent Technologies, Santa Clara, CA) and quantified using Kapa Library Quantification Kit (Kapa Biosystems, Wilmington, MA). Each library was sequenced in one lane of HiSeq4000 (Illumina, San Diego, CA) to achieve ~300 million reads following manufacturer's sequencing specification (10x Genomics). A balanced design was used for the experiment, where each genotype (Cop1wt/wt and Cop1fl/fl) had cells from one male and one female mouse.
attributes.tag: GEO Accession
attributes.value: GSM4433466