title: Illumina HiSeq 4000 paired end sequencing; RNA-Seq of bone marrow derived macophages (BMDMs) and brain tissue isolated from wild type and Irgm1 knockout mice
design: RNA-Seq of bone marrow derived macophages (BMDMs) and brain tissue isolated from wild type and Irgm1 knockout mice
library_construction_protocol: The bone marrow cells from wild type (Irgm1+/+) and knockout (Irgm1-/-) mice were isolated and differentiated into macrophages by standard procedure. Briefly, six to eight weeks old male C57BL/6 Irgm1+/+and Irgm1-/- mice were sacrificed by cervical dislocation, bone marrow cells from the tibia and femurs were flushed out in RPMI medium. Red blood cells were removed by cell lysis buffer containing (155 mM NH4Cl, 12 mM NaHCO3 and 0.1 mM EDTA). Bone marrow cells were differentiated in RPMI medium (10% FBS, 1mM sodium pyruvate and 0.05 M 2-mercaptoethanol) containing 20 ng/mL mouse M-CSF (Gibco #PMC2044) for 5 days. On every alternate day, media was replaced with fresh media containing M-CSF. Brain was excised from wild type and irgm1 knockout mice RNA was extracted from brain tissues and BMDMs of three different mice (three biological replicates) using RNeasy mini kit (QIAGEN, #74104). The quality and quantity of total RNA was checked using agarose gel and Qubit 3.0 After assessing the quality of RNA, 800-900 ng of total RNA was subjected to NEBNext® ultra™ directional RNA library prep kit for Illumina® (#E7420) using NEBNextPoly(A) mRNA magnetic isolation module (NEB #E7490). The QC of the prepared library was performed using High-Sensitivity Tape Station Kit (Agilent 2200, 5067-5585 (reagents) #5067-5584 ( screen tapes)) for fragment length distribution and Qubit dsDNA HS assay kit (Invitrogen, Q32851) for quantifying the library