GSM8734999: PMN_isotype_1; Homo sapiens; RNA-Seq
After activation, PMN were stained with PB-labeled anti-CD11b, FITC-labeled anti-CD66b, and APC-labeled anti-human CD62L (clone DREG-56, BioLegend). Activation and degranulation were determined as upregulation of CD11b or CD66b or as downregulation of CD62L. PMN activation or degranulation after stimulation with LPS or anti-TREM-1 (1C5) served as inclusion criteria, and PMN activation in the medium control group (preactivation) or after treatment with the isotype (4C9) were defined as exclusion criteria (33). Barcoded mRNA seq libraries were prepared from 200 ng total RNA. For library preparation at Novogene, the mRNA present in the total RNA sample was isolated with magnetic beads of oligos d(T)25. This method is known as polyA-tailed mRNAenrichment. Subsequently, mRNA was randomly fragmented and cDNA synthesis proceeds through the use of random hexamers and the reverse transcriptase enzyme. Once the synthesis of the first chain is finished, the second chain is synthesized with the addition of an Illumina buffer. With this and together with the presence of dNTPs, RNase H and polymerase I from E.coli, the second chain is obtained by Nick translation. The resulting products go through purification, end-repair, A-tailing and adapter ligation. Fragments of the appropriate size are enriched by PCR, where indexed P5 and P7 primers are introduced, and final products are purified. The library was checked with Qubit 2.0 and real-time PCR for quantification and bioanalyzer Agilent 2100 for size distribution detection. Quantified libraries have been pooled and sequenced on Illumina Novaseq 6000 platform, according to effective library concentration and data amount. Sequencing strategy was paired-end 150 cycles (PE150).