GSM4156074: AGC04CSF_90; Homo sapiens; RNA-Seq
We obtained four peritoneal ascites and one cerebrospinal fluid (AGC04CSF) aspirated for the treatment purpose. Suspending cells were collected by centrifuge. To construct reference M1 or M2 type macrophages, fresh PBMCs of two healthy donors were separated from whole blood samples by Ficoll-Paque. Only CD14+ monocytes were selected out using MACS human CD14 MicroBeads, Pre-Separation Filters, and LS columns as following the manufacture's recommend. To induce M0 macrophages, firstly isolated CD14+ monocytes were seeded into FBS coated 24-well plate with a density of 1 × 105 cells/cm2. Seeded cells were cultured 7 days in RPMI1640 media supplied with 20% of FBS and 100ng/ml of M-CSF. At day 7, M-CSF containing media were removed and appropriate stimulating media with 100ng/ml LPS and 20ng/ml IFN-γ for M1 or 20ng/ml IL-4 and 20ng/ml IL-10 for M2 were supplied. Fully differentiated M1 or M2 cells were collected by smooth scrapping after additional 48 hour of culture. Single-cell transcriptome data for AGC and M1/M2 macrophages were prepared using the C1 Single-Cell Auto Prep System following the manufacturer's instruction. Freshly prepared cell suspensions were loaded onto the C1 system and amplified cDNAs were obtained as the product. The microfluidic chip type used in the C1 system was determined by the size distribution and average cell size of each sample. We quantified amplified cDNAs using a Qubit® 2.0 Fluorometer then qualified using 2100 Bioanalyzer . Successfully amplified single-cell cDNAs (from 180 cells of AGC, 97 of M1 and 45 of M2) were respectively subjected to RNA sequencing. Sequencing libraries were constructed with the Nextera XT DNA Sample Prep Kit and sequenced by the HiSeq2500 system in 100-bp paired-end mode using the TruSeq Rapid PE Cluster kit and the TruSeq Rapid SBS kit.