GSM6172172: fl/fl Cre+ 24 h LPS rep1; Mus musculus; RNA-Seq
MHCII positive cells were immunomagnetically isolated (Miltenyi Biotec) and lysed (RLT PlusLysis Buffer; Qiagen, Hilden, Germany). RNA was purified with the RNeasy Plus Micro Kit as recom-mended by the manufacturer (Qiagen) and quantified using a Qubit 2.0 fluorometer (Invi-trogen). RNA quality was assessed on a Bioanalyzer 2100 using a RNA 6000 Pico chip (both from Agilent, Santa Clara, CA) RNA quality was assessed on a Bioanalyzer 2100 using a RNA 6000 Pico chip (both from Agilent, Santa Clara, CA). 10 ng of total RNA (integrity > 8) was used to generate barcoded mRNA-seq cDNA library using the NEBNext® Poly(A) mRNA Magnetic Isolation Module and NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (both from NEB, Ipswich, MA) with a final amplification of 15 PCR cycles. RNA was quantified using the Qubit HS assay kit (Thermo Fisher) and the library size distribution was determined using the Bioanalyzer HS DNA assay (Agilent). Afterwards, barcoded RNA-Seq libraries were clustered using the HiSeq® Rapid SR Cluster Kit v2 (Illumina, San Diego, CA) using 8pM and 59bps were sequenced on the Illumina HiSeq2500 using HiSeq® Rapid SBS Kit v2 (59 cycles).