title: GSM6640011: Treg, CD18wt, stimulated, biol rep3; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated using the RNeasy Plus mini kit (Qiagen, Hilden, Germany) as recommended and was quantified employing a Qubit 2.0 fluorometer (Thermo Fisher/Invitrogen, Waltham, MA). RNA quality was determined using a Bioanalyzer 2100 with a RNA 6000 Pico chip (both from Agilent, Santa Slara, 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. Afterwards, RNA was quantified using the Qubit HS assay kit (Thermo). The library size distribution was determined using the Bioanalyzer HS DNA assay (Agilent). Then, 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).