GSM9338230: Sting-deficient GM-BMCs treated with medium (control) for 6h (replicate 2); Mus musculus; RNA-Seq
Bone marrow cells were isolated from the legs of mice and cultured in RPMI 1640 (Nacalai Tesque, Kyoto, Japan) containing 10% foetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA) and 10 ng/ml of GM-CSF (PeproTech, Cranbury, NJ, USA). Red blood cells (RBCs) were lysed with RBC lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) before culture. After 4 days, additional GM-CSF (10 ng/ml) was added and used for the experiment on days 6–7. Cells were treated with HCQ or cGAMP for the indicated time periods. Transcriptome libraries were prepared from cell lysate resolved in lysis buffer. PolyA RNAs were isolated using Dynabeads M-270 Streptavidin (Thermo Fisher Scientific) conjugated with biotin-3' WTA-EcoP-dT25 according to the previous report with some modifications (GSE110711). To perform reverse transcription, beads were then suspended in 10 μL of RT mix [5× Superscript IV buffer (Thermo Fisher Scientific), 1 mM dNTP (Roche), 5 mM DTT (Thermo Fisher Scientific), 1 M betaine (Sigma), 6 mM MgCl2, 1 U/μL RNaseIn Plus Rnase Inhibitor (Promega), 10 U/μL Superscript IV (Thermo Fisher Scientific)], and incubated for 5 min at 35ºC, 30 min at 42ºC, and immediately cooled on ice. Beads were washed once with B&W-T buffer [5 mM Tris-HCl (pH 8.0) (Nippon Gene), 1 M NaCl (Merck), 1 mM EDTA (Nippon Gene), and 0.05% Tween-20 (Merck)], and once with Tris-HCl (pH 8.0). To digest the free primer, beads were suspended in 10 μL of Exo I mix [10× Exo I buffer (New England Biolabs), 2 U/μL Exonuclease I (New England Biolabs)], incubated for 30 min at 37ºC, and immediately cooled on ice. Beads were washed twice with B&W-T buffer and once with Tris-HCl (pH 8.0). To add polyC tail, beads were then suspended in 10 μL of polyC tailing mix [10× Thermopol buffer (New England Biolabs), 2 mM dCTP (Roche), 0.1 mM ddCTP (GE Healthcare), 1 mM CoCl2 (Roche), RNaseH (Invitrogen), 15 U/ul TdT enzyme (Roche)], and incubated for 30 min at 37ºC and immediately cooled on ice. Beads were washed once with B&W-T buffer and once with Tris-HCl (pH 8.0). To synthesize the 2nd strand, beads were then suspended in 10 μL of 2nd strand mix [1x KAPA Hifi Hotstart ReadyMix (KAPA Biosystems), 0.4 μM of primer (illumina-i7-9G)], and the thermal cycling was performed as follows: for 3 min at 95°C, 20 sec at 98°C, 16 cycles of 1 min at 47°C and 2 min at 72°C, followed by 5 min at 72°C, and hold at 4°C. To amplify the total cDNA, 10 μL of beads containing cDNA was added with 15 μL of first PCR mix [0.32 μM of primer (illumina-i7), 0.48 μM of primer (NH2-3' WTA), and 1x KAPA Hifi Hotstart ReadyMix], and the thermal cycling was performed as the following condition: for 3 min at 95°C, 12 cycles of 20 sec at 98°C, 15 sec at 65°C, and 5 min at 72°C, followed by 5 min at 72°C, and hold at 4°C. The first PCR products were purified using AmPure XP beads (Beckman-Coulter) at a 0.6:1 reagent-to-sample ratio and eluted with 25 μL of nuclease-free water. 6.3uL of purified first PCR product was mixed with the 8.7 μL of second PCR mix [0.4 μM of primers (illumina-i7, NH2-3' WTA), and 1× KAPA Hifi Hotstart ReadyMix], and the thermal cycling was performed as following condition: for 3 min at 95°C, 5 cycles of 20 sec at 98°C, 15 sec at 65°C, and 5 min at 72°C, followed by 5 min at 72°C, and hold at 4°C. The second PCR products were purified using AmPure XP beads at a 0.6:1 reagent-to-sample ratio and eluted with 15 μL of 10 mM Tris-HCl (pH 8.0). 100 ng of the whole-transcriptome library was subjected to fragmentation/end-repair/A-tailing using NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs) with some modifications. The thermal cycling was performed under the following conditions: for 7 min at 32ºC, 30 min at 65ºC, and hold at 4ºC. Then, 1.25 μL of 1.5 μM Illumina adapter was used for adapter ligation. Ligated products were purified by double size selection with 0.4× → 0.7× (final 1.1×) AmPure XP beads and eluted with 10 μL of nuclease-free water. The barcoding PCR was performed with 22.5 uL of barcoding mix [7.5 μL of the resulted eluates, 1 μM primers (ILMN_[UDI]_i5 and ILMN_[UDI]_i7), and 1× NEBNext Ultra II Q5 (New England Biolabs)], and the thermal cycling was performed as following condition: for 30 sec at 98°C, 9 cycles of 10 sec at 98°C and 75 sec at 65°C, followed by 5 min at 65°C, and hold at 4°C. The resulting products were purified twice by double size selection with 0.5× → 0.8× (final 1.3×) AmPure XP beads and eluted with 12 μL of 10 mM Tris-HCl, pH 8.0. The size distribution of amplified products was analyzed by the MultiNA system (Shimazu) with appropriate dilutions. Final transcriptome libraries, approximately 300 base pairs in length, were quantified using the KAPA Library Quantification Kit (KAPA Biosystems). Pooled libraries were sequenced by the Illumina Novaseq 6000 sequencer (Illumina).