GSM5668493: AA_LPS; Homo sapiens; RNA-Seq
RNA isolation was performed using the miRNeasy Mini kit (217004, Qiagen) according to the manufacturer's protocol including the DNase treatment. The RNA concentration was determined using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Waltham). cDNA libraries were prepared according to the CelSeq2 protocol (Hashimshony et al., 2012; Simmini et al., 2014) by Single Cell Discoveries, Utrecht (Muraro et al., 2016). Briefly, for each sample a custom-made primer was added to the RNA, denatured at 70 °C for 2 min, and immediately cooled down. mRNA was reverse transcribed into cDNA using clean-up beads (ThermoFisher Scientific, Ambion, Waltham, USA). The purified cDNA was transcribed in vitro to obtain amplified RNA using the MegaScript kit, which was followed by purification with Agencourt RNAclean XP (RNAse free) beads (Beckman Coulter). Amplified RNA was fragmented with fragmentation buffer and purified using the RNA cleanXP beads. Quality of the amplified RNA was determined with the RNA 6000 Pico chips (Agilent) on an Agilent 2100 bioanalyzer. Next, the amplified RNA was reverse transcribed and amplified by PCR. Libraries were labelled with a 4 bp unique molecular identifier (UMI) that was added to the primer. Samples from the different stimulation and control conditions were pooled in libraries for each donor (see Supplementary Table 1). The quality of the libraries was determined with the DNA High Sensitivity chips on an Agilent 2100 bioanalyzer (Agilent). Libraries were sequenced on the Illumina NextSeq 500 platform using paired-end sequencing (75 bp) with a depth of 10M reads per sample. Cel-seq2