title: KO3 GLUT1-low
organism: Mus musculus
description: Protocols: LLC tumor-bearing mice were sacrificed by cervical dislocation and tumors were harvested. Tumors were minced in RPMI medium containing 0.1% collagenase type I and 0.2% dispase type I and incubated in the same solution for 30 minutes at 37°C. The digested tissue was filtered using a 70 μm pore sized mesh and cells were centrifuged 5 min at 1000 rpm. Red blood cell lysis was performed by using Hybri-MaxTM (Sigma-Aldrich). For flow sorting, the myeloid cell population in the tumor single cell suspension was enriched by coating with CD11b-conjugated magnetic beads (MACS, Miltenyi Biotec) and separation through magnetic columns (MACS, Miltenyi Biotec). Cells were resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) and incubated for 15 minutes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb (BD-pharmingen) and stained with the following antibodies for 20 minutes at 4 °C: anti-F4/80 and anti-GLUT1. Cells were subsequently washed and resuspended in cold FACS buffer before flow sorting by a FACS Aria (BD Biosciences). REDD1 KO mice were generated by Lexicon Corp (USA) exclusively for Quark Pharmaceuticals Inc and are the property of Quark Pharmaceuticals Inc. WT mice were littermate controls. 5-6 weeks old C57BL/6N recipient mice were irradiated with 9.5 Gy. Subsequently, 107 bone marrow cells from the appropriate genotype were injected intravenously via the tail vein. Tumor experiments were initiated 5 weeks after bone marrow reconstitution. : 1 x 106 Lewis lung carcinoma (LLC) adherent growing murine cells were injected subcutaneously at the right side of the mouse in a volume of 200 μl of PBS. Animals were killed 3 weeks after tumor cell injection, and the tumors were processed for FACS sorting immediately. RNeasy microkit from Qiagen RNA concentration and purity from freshly sorted TAMs were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 100 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version 15031047 Rev.E) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichment PCR of 15 cycles was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library.
attributes.tag: ENA first public
attributes.value: 2018-01-01
attributes.tag: ENA last update
attributes.value: 2016-08-12
attributes.tag: External Id
attributes.value: SAMEA4378829
attributes.tag: INSDC center alias
attributes.value: VIB - KULEUVEN
attributes.tag: INSDC center name
attributes.value: VIB - KULEUVEN
attributes.tag: INSDC first public
attributes.value: 2018-01-01T17:02:29Z
attributes.tag: INSDC last update
attributes.value: 2016-08-12T16:35:08Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-5032:KO3 GLUT1-low
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: CD11b+ F4/80+ macrophage
attributes.tag: common name
attributes.value: house mouse
attributes.tag: disease
attributes.value: Lewis lung carcinoma
attributes.tag: genotype
attributes.value: Ddit4 knockout
attributes.tag: phenotype
attributes.value: low GLUT1 expression
attributes.tag: sample name
attributes.value: E-MTAB-5032:KO3 GLUT1-low
attributes.tag: strain
attributes.value: C57BL/6N