title: GSM6138964: chimera_5w alveolar macrophages CD45.2-CD11cCre Cebpb KO rep4 [lung_2.2_5w_4]; Mus musculus; RNA-Seq
library_construction_protocol: lung digestion in RPMI complete with 1mM collagenase IV and 2mg/ml DNase I at 37°C for 30 min mRNA was subjected to reverse transcription using an Affinity Script cDNA Synthesis Kit (Agilent) and MARS-seq barcoded RT primers. Samples were analyzed by qPCR and up to 8 samples with similar Ct values were combined. Pooled samples were then treated with Exonuclease I (New England BioLabs (NEB)) and incubated at 37°C for 30min and 80°C for 10min followed by a 1.2X AMPure XP beads (Beckman Coulter) cleanup. Second strand synthesis of the cDNA was performed using a second strand synthesis kit (NEB) at 16°C for 2h and samples were cleaned with 1.4X volumes of AMPure XP beads. Afterwards samples were subjected to in vitro transcription (IVT) with a HiScribe T7 RNA Polymerase kit (NEB) at 37°C for 13-16h. The DNA template was removed by Turbo DNase I (Life Technologies) treatment at 37°C for 15min and the samples were cleaned with 1.2X volumes of AMPure XP beads. Subsequently, the RNA was fragmented at 70°C using RNA fragmentation buffer (Invitrogen), after 3min the reaction was stopped with Stop buffer (Invitrogen) and samples were purified by 2X SPRI cleanup. Afterwards T4 RNA ligase (NEB) was used to ligate the fragmented RNA to the MARS-seq ligation adapter (22°C 2h) and samples were purified with a 1.5X AMPure XP beads cleanup. In a second reverse transcription reaction (reaction volume 20µl) using the MARS-seq RT2 primer and an Affinity Script cDNA Synthesis Kit (Agilent Technologies) the RNA was transcribed into cDNA followed by another 1.5X AMPure XP beads purification. As final step the library was amplified in a nested PCR with P5_Rd1 and P7_Rd2 primers and Kapa HiFi Hotstart ready mix (Kapa Biosystems) and purified with a 0.7X AMPure XP beads cleanup. The fragment size of the final libraries was measured using a TapeStation (Agilent Technologies) and library concentrations were quantified with a Qubit fluorometer (Life Technologies). The samples were sequenced using a NextSeq 500 system (Illumina). RNA-Seq:Cells for RNA-Seq or ATAC-Seq were isolated by FACS. Cells for RNA-Seq (500-20,000 sorted cells) were lysed with 50-200µl of lysis/binding buffer (Life Technologies), frozen on dry ice and stored at -80°C until further use. The mRNA was purified using a Dynabeads mRNA DIRECT Purification Kit (Life Technologies) according to the manufacturer's guidelines. Reverse transcription was performed with MARS-seq barcoded RT primers and the Affinity Script cDNA Synthesis Kit (Agilent) in a 10µl reaction volume. RNA-Seq:mRNA was subjected to reverse transcription using an Affinity Script cDNA Synthesis Kit (Agilent) and MARS-seq barcoded RT primers. Samples were analyzed by qPCR and up to 8 samples with similar Ct values were combined. Pooled samples were then treated with Exonuclease I (New England BioLabs (NEB)) and incubated at 37°C for 30min and 80°C for 10min followed by a 1.2X AMPure XP beads (Beckman Coulter) cleanup. Second strand synthesis of the cDNA was performed using a second strand synthesis kit (NEB) at 16°C for 2h and samples were cleaned with 1.4X volumes of AMPure XP beads. Afterwards samples were subjected to in vitro transcription (IVT) with a HiScribe T7 RNA Polymerase kit (NEB) at 37°C for 13-16h. The DNA template was removed by Turbo DNase I (Life Technologies) treatment at 37°C for 15min and the samples were cleaned with 1.2X volumes of AMPure XP beads. Subsequently, the RNA was fragmented at 70°C using RNA fragmentation buffer (Invitrogen), after 3min the reaction was stopped with Stop buffer (Invitrogen) and samples were purified by 2X SPRI cleanup. Afterwards T4 RNA ligase (NEB) was used to ligate the fragmented RNA to the MARS-seq ligation adapter (22°C 2h) and samples were purified with a 1.5X AMPure XP beads cleanup. In a second reverse transcription reaction (reaction volume 20µl) using the MARS-seq RT2 primer and an Affinity Script cDNA Synthesis Kit (Agilent Technologies) the RNA was transcribed into cDNA followed by another 1.5X AMPure XP beads purification. As final step the library was amplified in a nested PCR with P5_Rd1 and P7_Rd2 primers and Kapa HiFi Hotstart ready mix (Kapa Biosystems) and purified with a 0.7X AMPure XP beads cleanup. The fragment size of the final libraries was measured using a TapeStation (Agilent Technologies) and library concentrations were quantified with a Qubit fluorometer (Life Technologies). The samples were sequenced using a NextSeq 500 system (Illumina). ATAC-seq: For the preparation of ATAC-seq libraries 15,000-20,000 cells were used as described earlier (Lara-Astiaso et al., 2014; Buenrostro et al., 2013). Briefly, nuclei were isolated by lysis with cold lysis buffer (10mM Tris-HCl pH7.4, 10mM MgCl2, 0.1% Igepal CA-630) and centrifuged for 25min at 500xg and 4°C with low acceleration and brake using a swing-out rotor. The supernatant was carefully removed and nuclei were resuspended in 25μl reaction buffer containing 2μl of Tn5 transposase and 12.5μl of TD buffer (Nextera DNA library preparation kit; Illumina). The mix was incubated at 37°C for 1hr. Afterwards, the DNA was purified by adding 5μl of cleanup buffer (900mM NaCl, 30mM EDTA), 2μl of proteinase K (NEB) and 2μl of 5% SDS. After an incubation phase of 30min at 40°C, the tagmented DNA was enriched using AMPure XP beads (Beckman Coulter). The DNA was PCR-amplified with indexed primers and Kapa HiFi Hotstart ready mix (Kapa Biosystems). After the PCR, tagmented DNA fragments were selected for fragments smaller than 600bp and purified using AMPure XP beads. Libraries were subjected to a second PCR amplification with Kapa HiFi Hotstart ready mix (Kapa Biosystems), indexing primers and an appropriate number of reaction cycles depending on the library concentration. Library sizes were determined using a TapeStation (Agilent Technologies) and measurement of DNA concentration was performed with a Qubit fluorometer (Life Technologies). Libraries where sequenced with an average of 20 million reads per sample on a NextSeq500 system (Illumina). ChIP-seq: Preparation of ChIPmentation samples was performed according to Schmidl et al., 2015. 2M AMs were BAL-isolated from C57BL/6 mice and fixed in a 1% PFA solution (in 10% FBS/PBS). The fixation reaction was quenched on ice by addition of glycine (125mM final concentration) and the samples were centrifuged at 300xg for 5min at 4°C. Subsequently cell pellets were washed twice with cold PBS and 1X Protease Inhibitor (Roche), snap-frozen in liquid nitrogen and stored at -80°C until further processing. The samples were thawed on ice and resuspended in Chromatin Prep Buffer (High Sensitivity Chromatin Preparation kit, Active Motif) with 1X Protease Inhibitor (Roche) before nuclei were released using a dounce homogenizer with a tight pestel (Active Motif) and centrifuged at 4°C and 1250xg for 5min. Nuclei were then resuspended in cold Sonication Buffer (0.25% SDS, 10mM Tris-HCl pH 8.0, 2mM EDTA, 1X Protease Inhibitor) and pipetted to facilitate nuclei disruption. The chromatin was sonicated using a Diagenode Bioruptor (12 cycles: 20sec on, 30sec off) to achieve a fragment size ranging from 200-500bp. 1% of the sonicated chromatin was used for the INPUT sample, the rest was incubated overnight with pre-washed A Dynabeads (Thermofisher; previously blocked with 0.1% BSA) and 4μg of anti-C/EBPβ antibody (C-19; Santa Cruz Biotechnology). The following day, the beads were washed twice with RIPA-LS (10mM Tris-HCl pH8.0, 1mM EDTA pH8.0, 140mM NaCl, 0.1% SDS, 0.1% Na-Deoxycolate, 1% Triton X-100, 1X Protease Inhibitor), twice with RIPA-HS (10mM Tris-HCl pH8.0, 1mM EDTA pH8.0, 500mM NaCl, 0.1% SDS, 0.1% Na-Deoxycolate, 1% Triton X-100, 1X Protease Inhibitor), twice with RIPA-LiCl (10mM Tris-HCl pH8.0, 1mM EDTA pH8.0, 250mM LiCl, 0.1% SDS, 0.5% Na-Deoxycolate, 1% Triton X-100, 1X Protease Inhibitor), twice with 10mM Tris-HCl pH 8.0 and finally resuspended and incubated in the Tagmentation Solution (0.25% Tagmentation Buffer, 2mM Tn5 (Illumina)) for 2min at 37°C. The tagmentation reaction was stopped on ice by adding cold RIPA-LS. Later, beads were washed twice in RIPA-LS, twice in 1X TE and finally resuspended in ChIP Elution Buffer (10mM Tris-HCl pH8.0, 5mM EDTA pH8.0, 300mM NaCl, 0.4% SDS). Samples were de-crosslinked overnight and purified on the following day using AMPure XP beads (NEB). Libraries were amplified using KAPA HiFi Hotstart ready mix (Kapa Biosystems) and indexing primers published by Schmidl et al. 2015 (22) and finally purified using AMPure XP beads (NEB). Library sizes were determined using a TapeStation (Agilent Technologies) and measurement of DNA concentration was performed with a Qubit fluorometer (Life Technologies). Libraries were sequenced with an average of 30 million reads per sample on a NextSeq500 system (Illumina).