title: GSM4066498: IM RNA-seq C57BL6J rep2; Mus musculus; RNA-Seq
library_construction_protocol: Lungs were incubated at 37oC for 15 minutes while on a rotator. Red blood cells were lysed using RBC lysis buffer (eBioscience) for a 5 minute incubation. The obtained cell suspension was filtered using a 70 micron filter, washed and cells were resuspended in PBS. Cells were then incubated with anti-Fc blocking mAb (anti CD16/32, clone 93, BioLegend) and viability dye Zombie Aqua (Biolegend) for 10 min. After this, cells where stained with an antibody cocktail containing APC Cy7-conjugated CD45, PE-conjugated F4/80, BV605-conjugated CD11c, v450-conjugated CD11b, PE Cy7-conjugated CD64, AF700-conjufgated Ly6c, FITC-conjugated Ly6g, AF647-conjugated SiglecF mAb for 30 minutes. All labeled cells were passed through a FACS Aria II flow cytometer (BD Biosciences) and after proper gating 4 subsets of lung MP were sorted RNA-seq: PolyA enriched mRNA was fragmented, in 2x Superscript III first-strand buffer with 10mM DTT (Invitrogen), by incubation at 94°C for 9 minutes, then immediately chilled on ice before the next step. The 10 µL of fragmented mRNA, 0.5 µL of Random primer (Invitrogen), 0.5 µL of Oligo dT primer (Invitrogen), 0.5 µL of SUPERase-In (Ambion), 1 µL of dNTPs (10 mM) and 1 µL of DTT (10 mM) were heated at 50°C for three minutes. At the end of incubation, 5.8 µL of water, 1 µL of DTT (100 mM), 0.1 µL Actinomycin D (2 µg/µL), 0.2 µL of 1% Tween-20 (Sigma) and 0.2 µL of Superscript III (Invitrogen) were added and incubated in a PCR machine using the following conditions: 25°C for 10 minutes, 50°C for 50 minutes, and a 4°C hold. The product was then purified with RNAClean XP beads according to manufacture's instruction and eluted with 10 µL nuclease-free water. The RNA/cDNA double-stranded hybrid was then added to 1.5 µL of Blue Buffer (Enzymatics), 1.1 µL of dUTP mix (10 mM dATP, dCTP, dGTP and 20 mM dUTP), 0.2 µL of RNAse H (5 U/µL), 1.05 µL of water, 1 µL of DNA polymerase I (Enzymatics) and 0.15 µL of 1% Tween-20. The mixture was incubated at 16°C for 1 hour. The resulting dUTP-marked dsDNA was purified using 28 µL of Sera-Mag Speedbeads (Thermo Fisher Scientific), diluted with 20% PEG8000, 2.5M NaCl to final of 13% PEG, eluted with 40 µL EB buffer (10 mM Tris-Cl, pH 8.5) and frozen -80°C. The purified dsDNA (40 µL) underwent end repair by blunting, A-tailing and adapter ligation as previously described44 using barcoded adapters (NextFlex, Bioo Scientific). Libraries were PCR-amplified for 14-16 cycles, size selected using 10% PAGE/TBE gels, eluted and quantified with Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), ATAC-seq: Sorted cells in FACS Buffer were pelleted by centrifugation at 400 RCF for 6 minutes at 4oC in a pre-cooled fixed-angle microfuge. Supernatant was carefully removed and cell pellet was resuspend in 47.5 ul lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630), 2.5ul of TDE1 (Cat# FC-121-1030, Illumina) by pipetting. Transposition reactions were incubated at 37°C for 30 minutes. Transposed DNA was purified using a Zymo ChIP DNA Clean & Concentrator kit (Cat# D5205) and purified DNA was eluted in 10 ul elution buffer (10 mM Tris-HCl, pH 8). Transposed fragments were amplified and purified as described previously (Buenostro et al) with modified primers.
attributes.tag: GEO Accession
attributes.value: GSM4066498