title: GSM6051647: PRMT1Ifl/fl microglia CPZ diet_CD11c_high; Mus musculus; RNA-Seq
library_construction_protocol: Brains were digested, and myelin were removed using 30% percoll gradient. Microglia was sorted using CD45 and CD11B surface markers. RNA in 2X SuperScript III buffer was incubated for 1 min at 50 C on a PCR cycler with 2.5 ul of the following mix: 1.5 ug Random Primer (Life Technologies 48190-011), 10 M Oligo d(T) (Life Technologies 18418020), 10 units SUPERase-In (Ambion AM2696), 4 mM dNTP mix (Life Technologies 18427088), in water. Samples were then immediately placed on ice for 5 min. First-strand synthesis was then performed by incubation at 25 C for 10 min and 50 C for 50 min on a PCR cycler with 7.6 ul of the following mix: 0.2 ug actinomycin (Sigma A1410), 13.15 mM DTT (Life Technologies), 0.026% Tween-20, 100 units SuperScript III (Life Technologies kit 18080-044), in water. After incubation, RNA/DNA complexes were isolated by adding 36 ul of Agencourt RNAClean XP beads (Beckman Coulter A63987) and incubated for 10 min at RT and then 10 min on ice. Samples were then placed on a magnet and beads were washed twice with 150 ul of 75% EtOH. Following washings, beads were air-dried for 10-12 min and eluted with 10 ul of water. Second-strand synthesis was then performed. RNA/DNA samples in 10 ul of water were incubated for 2.5 hours at 16 C with 5 ul of the following mix: 3x Blue Buffer (Enzymatics), 1.0 ul PCR mix (Affymetrix 77330), 2.0 mM dUTP (Affymetrix 77206), 1 unit RNAseH (Ezymatics Y9220L), 10 units DNA Polymerase I (Enzymatics P7050L), 0.03% Tween-20, in water). DNA was then purified by addition of 1.5 ul Sera-Mag SpeedBeads (Thermo Scientific, 651520505025), resuspended in 30 ul 20% PEG 8000/2.5 M NaCl, incubated at RT for 15 min and placed on a magnet for two rounds of bead washing with 80% EtOH. Beads were then air dried for 10-12 min and DNA was eluted from the beads by adding 40 ul of water. Supernatant was then collected on a magnet and placed on ice or stored at -20C until DNA blunting, poly(A)-tailing, and adapter ligation. Ethanol-precipitated with glycogen and dissolved in water at 9 μM) were heat-denatured together with the RNA for 2 min at 70°C, and ligated with 100 U truncated T4 RNA ligase 2 K227Q (NEB) in 10 μl 1x T4 RNA ligase buffer without ATP, containing 10 U SUPERase-In and 15% PEG8000 for 2 hr at 16°C. To reduce adaptor dimer formation, 0.5 μl 10 μM MPX_ RT primer 5′-GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T-3′ (IDT, desalted) was added and annealed to the ligation product by incubating at 75°C for 2 min, then 37°C for 30 min, then 25°C for 15 min. Finally, 0.5 μl 5 μM hybrid DNA/RNA sRNA5′h adaptor 5′-GTT CAG AGT TCT ACA rGrUrC rCrGrA rCrGrA rUrC-3′ (IDT) were ligated to previously capped RNA 5′ ends by adding 2 μl T4 RNA ligase buffer, 6 μl 50% PEG8000 (15% final), 1 μl 10 mM ATP, 9.5 μl water and 0.5 μl (5 U) T4 RNA ligase 1 for 90 min at 20°C. To 15 μl ligation reaction, an additional 0.5 μl 10 μM MPX_ RT primer were added, reactions were denatured at to 70°C for 1 min, then placed on ice. RNA was reverse-transcribed by adding 3 μl 10x first strand buffer, 4.5 μl water, 1.5 μl 10 mM dNTP, 3 μl 0.1 M DTT, 1.5 μl RNaseOUT and 1 μl Superscript III reverse transcriptase (Invitrogen), and incubating for 30 min at 50°C. Complementary DNA was isolated with Sera-Mag SpeedBeads (Thermo Scientific, 6515205050250) in 20% PEG/2.5M NaCl. and dissolved in 40 μl TET. RNA-seq libraries were digested by 30 min of incubation at 37C with Uracil DNA Glycosylase (final concentration of 0.134 units per ul of library volume; UDG, Enzymatics G5010L) to generate strand-specific libraries. Libraries were PCR-amplified for 12-15 cycles and size selected for fragments (200-500 for RNA-seq) by gel extraction (10% TBE gels, Life Technologies EC62752BOX).  RNA-seq librairies were single-end sequenced for 76 cycles on an Illumina HiSeq 4000 (Illumina, San Diego, CA) according to manufacturer's instruction.