title: GSM5728576: Control macrophages [control_ix2]; Homo sapiens; RNA-Seq
library_construction_protocol: After the treatment cells were washed with PBS and detached with TrypLE reagent after 15 min incubation at cell culture incubator (37 °C, 5% CO2). After centrifugation cells were washed twice with 1x RNase-free PBS (cat#AM9624, Invitrogen). After final centrifugation cells were resuspended in RNase-free PBS and kept on ice till cell preparation for RNA sequencing analysis. Single-cell RNA sequencing (scRNAseq) was performed using a modified version of the inDrops method (Zilionis et al. 2017, Klein et al. 2015). While the original version is based on the CEL-Seq protocol (Hashimshony et al., 2012) that involves linear cDNA amplification by in vitro transcription, the modified version relies on template switching and cDNA amplification by PCR, as in the Smart-seq protocol (Ramsköld,2012). First, single cell transcriptomes were barcoded in 1-nl droplets by co-encapsulating: i) barcoding hydrogel beads; ii) the reverse-transcription/lysis (RT/lysis) mix; and iii) the cell suspension. Cell encapsulation was performed using the microfluidic device described in Zilionis et al. 2017 on an Onyx platform (Droplet Genomics). Maxima H- minus (ThermoFisher, cat. no. EP0751) was used for reverse-transcription with template switching. RT was performed for 1h at 42 ºC followed by heat inactivation for 5 min at 85 ºC. The emulsion was then broken and the pooled material was taken through library preparation for Illumina sequencing, which involves the following steps: i) cDNA amplification (Terra PCR direct Polymerase, Takara, cat. no. 639270), ii) fragmentation and adapter ligation (NEBNext® UltraTM II FS DNA Library Prep Kit for Illumina, NEB, cat. no. E7805S), and iii) indexing PCR (KAPA HiFi HotStart ReadyMix PCR Kit, Roche, cat. no. KK2601). Sequencing was performed on a single Illumina NextSeq run (NextSeq 500/550 High Output Kit v2.5 (75 Cycles), Illumina, cat. no. 20024906).