title: GSM8763468: KLF2 KO BMM, day 0, rep 2; Mus musculus; RNA-Seq
library_construction_protocol: extraction protocol, Bone Marrow Macrophage culture and transfer On Day -7, femur, tibia and iliac bones from LysMCre/+Klf2fl/fl, LysM+/+Klf2fl/fl mice were flushed with RPMI (Gibco) and red blood cells were lysed with ACK buffer (Gibco). Cells were counted and 1M cells were plated in 10cm non-TC treated plates in 8 mL RPMI with 10mM HEPES (Gibco), 1mM Sodium Pyruvate (Gibco), 2mM Glutamax (Gibco), 100U/100µg/mLPen/Strep (Gibco), 1.7µL ß-mercaptoethanol (Fisher), 10% FCS (Gibco or Omega) (RP10+), 10% titrated M-CSF 3T3-cell conditioned laboratory-made media. On Day -3, BMM were given an additional 8mL RP10+ with 10% M-CSF. On Day -1, B6.SJL recipient mice were anesthetized with 90mg/kg ketamine + 5mg/kg xyalzine i.p. Upon achieving the appropriate anesthetic level, 5mL of warm PBS was injected slowly into the peritoneal cavity with a 23 gauge needle. Mice were gently rocked and then PBS was recovered with a 21 gauge needle. Mice were kept warm during the duration of the anesthesia and monitored until recovered. On Day 0, BMM were harvested by incubating in 5mL 4mM ETDA in PBS for 20 min in a 37 deg C incubator.  ETDA was quenched by the addition of 7mL RP10+. Cells were counted and resuspended to inject 1x106 cells per recipient mouse in 100µL of RPMI i.p. Cells were harvested by lavage on day 7, stained and sorted on live, single CD45+ cells with a BD FACS Aria Fusion (BD Biosciences). TRIzol LS (Thermo) was added to sorted cells and frozen at -80˚C for RNA extraction and library generation for RNAseq. extraction protocol, Bone Marrow Macrophage culture and transduction. On Day -1, 2.5x106 293T-GP2 retroviral producer cells were plated in 10mL DMEM with 10mM HEPES (Gibco), 1mM Sodium Pyruvate (Gibco), 2mM Glutamax (Gibco), 100U/100µg/mL Pen/Strep (Gibco), 1.7µL B-mercaptoehtanol(Fisher), 10% FCS (Gibco or Omega) (DM10+) in 10cm TC treated plates. On Day 0, 293T-GP2s (Clontech/Takara, RRID:CVCL_WI48) were transfected with 100ng MSCV plasmid (MSCV multiple cloning site IRES puroR 2A mCherry or GFP) and 10ng pVSVg plasmid (Addgene 8454) with 3µL/mL Lipofectamine 2000 (Thermo). Also on Day 0, femur, tibia and iliac bones from C57BL/6 mice were flushed with RPMI (Gibco) and red blood cells were lysed with ACK buffer (Gibco). Cells were counted and 4x106 cells were plated in 10 cm non-TC treated plates in 8 mL RPMI with 10mM HEPES (Gibco), 1mM Sodium Pyruvate (Gibco), 2mM Glutamax (Gibco), 100U/100µg/mL Penicillin/Streptomycin (Gibco), 1.7 µL B-mercaptoethanol (Fisher), 10% FCS (Gibco or Omega) (RP10+), and 10% titrated M-CSF 3T3-cell conditioned laboratory-made media. On Day 1, media was replaced on the 293T-GP2 to 8 mL. On Day 2 viral supernatants were harvested from 293T-GP2s and filtered through 0.45µM PES syringe filter. Bone marrow cultures were harvested, resuspended in 293T-GP2 supernatant supplemented with 10% M-CSF 3T3 cell media and returned to the same 10 cm plate. Media was replaced on 293T-GP2s for repeating of viral infections on Day 3. On Day 4, bone marrow cells are harvested out of plates and resuspended in 8 mL RP10+ with 10% M-CSF 3T3 supe with 5 µg/mL puromycin. Media was again replaced on Day 7 with fresh RP10+ with 10% M-CSF 3T3 supe. Cells were harvested on day 8-10 by incubating in 5mL 4mM ETDA in PBS for 20 min in a 37˚C incubator.  ETDA was quenched by the addition of 7mL RP10+. Cells were counted and 1x105 cells were resuspended in TRIzol (Thermo) and stored at -80˚C for subsequent RNA isolation. extraction protocol, Whole Lung Prep. The lungs were harvested and homogenized into a single-cell suspension with C tubes on a GentleMacs (Milltenyi) with appropriate tissue programs in 0.07mg/mL LiberaseTM and 45µg/mL DNaseI in 2mL HBSS with Ca/Mg. Tissues were incubated 20min at 37˚C and then finished on the GentleMacs. Enzymes were quenched with PBS with 2mM EDTA and 2% FCS on ice and then red blood cells were lysed with room-temperature ACK lysis buffer (Gibco) and passed through a 100μm cell strainer. Cells were sorted for live, single, CD45+CD11b-CD11c+ with a BD FACS Aria. Total RNA was isolated from cells using TRIzol or TRIzol LS reagent (Invitrogen). Chloroform extraction was performed according to the manufacturer's protocol. RNA was recovered from the aqueous layer using RNA Clean and Concentrator-5 kit (Zymo Research). RNA was quantified with a Qubit (Invitrogen) and the integrity was evaluated with Agilent Bioanalyzer 2100 (Agilent Technologies). Libraries were prepared using the SMART-seq2 protocol (Picelli et al., 2014). For BMM overexpressing transcription factors, samples were generated from two independent BMM transductions, but RNA was harvested, libraries were generated, and sequencing was all performed concurrently. Samples were sequenced on an Illumina NovaSeq sequencer using paired-end 50 base pair reads.