title: GSM8994519: 4D RA scRNAseq Ezh2 inhibitor GSK126 G2; Mus musculus; RNA-Seq
library_construction_protocol: For ChIP: cells were lysed, treated with MNAse, and histone-DNA complexes were isolated with antibody. For bulk RNA:mRNA extraction was performed using Invitrogen Dynabeads mRNA DIRECT Purification Kit (61011) according to the manufacturer's protocol. For scRNA: cells were collected into reverse transcription/lysis buffer mix. For ChIP libraries: 10ng DNA samples were converted to blunt ended, phosphorylated DNA using the End-It DNA End-Repair Kit (Lucigen). DNA was purified using ratio of 1:1.8 sample to AMPure XP beads. Adenosine was added to the 3' end of the DNA fragments using Klenow (3'-5' exo-) (New England Biolabs). DNA was purified using ratio of 1:1.6 sample to AMPure XP beads. Adapters containing Illumina library indices for multiplexing were 20 Article Title added (P7-index-PE2 and P5-PE1 long primers) by ligation using DNA ligase (New England Biolabs). DNA was purified using ratio of 1:1.2 sample to AMPure XP beads. DNA samples were amplified using KAPA HiFi HotStart (Roche) with 25μM primers (P7 and P5-PE1 short primers). Amplified libraries were purified using 0.7x reaction volume of AMPure XP beads and eluted in 22μℓ 10mM Tris-HCl [pH 8]. Resulting libraries were run in a 2% agarose gel, and the desired 250–600 bp DNA library fragments were selected and isolated using the PureLink DNA gel extraction kit (Invitrogen). For bulk RNA libraries: 500ng mRNA samples were fragmented using RNA Fragmentation Reagents (Thermo, AM8740) by combining 9μl of sample with 1μl of RNA fragmentation reagent and incubating at 70 °C for 2 min. The sample was transferred to ice, and 40μl fragmentation stop mix containing 5μl fragmentation stop solution and 35μl 10mM Tris- HCl [pH 8] was added. Fragmented RNA was purified with 1.5x reaction volume of AMPure XP beads and eluted in 10μl 10mM Tris- HCl [pH 8]. The fragmented RNA was reverse transcribed using an oligo dT Primer as follows: First, 10μl RNA was mixed with 2μl of 50μM oligo dT primer (IDT) and 1μl of 25mM dNTP mix (NEB). The sample was incubated for 5 min at 65 °C and transferred to ice. The following components were added to the reaction to a total volume of 20μl: 4μl of 5x First-Strand SuperScript III buffer (Thermo), 2μl of 0.1M DTT (Thermo), 1μl murine RNase inhibitor (NEB), and 1μl of 200 U/μl Super-Script III reverse transcriptase (Thermo) and the sample was incubated for 1 hour at 50 °C 15 minutes at 70 °C. cDNA was purified with 1.5x reaction volume of AMPure XP beads and eluted in 17μl 10mM Tris- HCl [pH 8]. For second-strand synthesis, 17μl were combined with 2μl second-strand synthesis (SSS) buffer and 1μl of SSS enzyme mix from the NEBNext mRNA Second Strand Synthesis Module (NEB) and incubated at 16 °C for 2.5 h, followed by 20 min at 65 °C. cDNA was purified with 1.2x reaction volume of AMPure XP beads and eluted in 14μl 10mM Tris- HCl [pH 8]. DNA samples were converted to blunt-ended, phosphorylated DNA using the End-It DNA End-Repair Kit (Lucigen). DNA was purified using 1.8x reaction volume of AMPure XP beads. Adenosine was added to the 3' end of the DNA fragments using Klenow (3'-5' exo-) (New England Biolabs). DNA was purified using 1.6x reaction volume of AMPure XP beads. Adapters containing Illumina library indices for multiplexing (P7-index-PE2 and P5-PE1 long primers) were added by ligation using DNA ligase (New England Biolabs). DNA was purified using 0.7x reaction volume of AMPure XP beads. The DNA samples were amplified using KAPA HiFi HotStart (Roche) with 25μM primers (P7 and P5-PE1 short primers). Amplified libraries were purified using 0.7x reaction volume of AMPure XP beads and eluted in 22μl 10mM Tris-HCl [pH 8]. Resulting libraries were run in a 2% agarose gel, and the desired 250–600 bp DNA library fragments were selected and isolated using a PureLink DNA gel extraction kit (Invitrogen). For scRNA libraries: The reverse transcription/lysis mix consisted of 180 μL of 5X First-Strand buffer (Invitrogen), 27 μL 10% (v/v) IGEPAL CA-630 (Sigma), 18 μL 25 mM dNTPs (NEB), 30 μL 0.1 M DTT (Invitrogen), 45 μL 1 M Tris-HCl [pH 8.0] (Biological Industries), 30 μL murine RNase inhibitor (NEB), 45 μL SuperScript III reverse transcriptase (200 U/μL, Invitrogen), and 75 μL nuclease-free water (Biological Industries) in a total volume 450 μL. The carrier oil was HFE-7500 with 3% (w/w) fluorosurfactant (RAN Biotechnologies. The collection tubes were exposed to 6.5 J/cm2 of 365-nm UV light for 10 min to release photocleavable barcoding primers from the barcoding beads. Next, the collection tubes containing the UV-exposed emulsions were transferred to a reverse transcription reaction at 50°C for 2 h followed by 15 min at 70°C. Each sample was demulsified by addition of 50 μL perfluoro-1-octanol (Sigma) to release the barcoded cDNA from the droplets. To remove unused primers and primer dimers, the upper, aqueous phase containing the barcoded cDNA (~50 μL) was combined with 50 μL digestion mix containing 5 μL ExoI enzyme (NEB), 5 μL HinFI enzyme (NEB), 5 μL ExoI buffer (NEB), 5 μL CutSmart buffer (NEB), and 30 μL of nuclease-free water and incubated for 1 h at 37°C and 10 min at 80°C. The reaction product was purified with 1.5x reaction volume of AMPure XP beads and eluted in 13.5 μL TE buffer (10 mM Tris–HCl [pH 8.0], 0.1 mM EDTA). For second-strand synthesis, 13.5 μL digestion reaction product was combined with 1.5 μL second-strand synthesis (SSS) buffer and 1 μL of SSS enzyme mix from the NEBNext mRNA Second Strand Synthesis Module (NEB) and incubated at 16°C for 2.5 h, followed by 20 min at 65°C. For in vitro transcription, SSS reaction products (16 μL) were combined with 24 μL HiScribe T7 High Yield RNA Synthesis Kit (NEB) reagent mix containing 4 μL T7 Buffer, 4 μL ATP, 4 μL CTP, 4 μL GTP, 4 μL UTP, and 4 μL T7 enzyme mix. The reaction was incubated at 37°C for 13 h, and the resulting RNA was purified with 1.3x reaction volume of AMPure XP beads and eluted with 20 μL TE buffer. RNA was fragmented using RNA fragmentation kit (Ambion), and 9 μL of this sample was combined with 1 μL of RNA fragmentation reagent and incubated at 70°C for 2 min. The sample was transferred to ice, and 40 μL fragmentation stop mix containing 5 μL fragmentation stop solution and 35 μL TE buffer was added. Fragmented RNA was purified with 1.3x reaction volume of AMPure XP beads and eluted in 10 μL TE buffer. The resulting amplified and fragmented RNA was reverse transcribed using a random hexamer primer as follows: First, 10 μL RNA was mixed with 2 μL of 100 μM PvG748-SBS12-RT random hexamer primer (IDT) and 1 μL of 10 mM dNTP mix (NEB). The sample was incubated for 3 min at 65°C and transferred to ice. The following components were added to the reaction to a total volume of 20 μL: 4 μL of 5x First-Strand SuperScript III buffer (Invitrogen), 1 μL of 0.1 M DTT (Invitrogen), 1 μL murine RNase inhibitor (NEB), and 1 μL of 200 U/μL SuperScript III reverse transcriptase (Invitrogen). The resulting cDNA was purified with 1.2x reaction volume of AMPure XP beads and eluted in 11.5 μL TE buffer. The resulting libraries were PCR amplified using standard PE1/PE2 full-length primer mix containing Illumina library indices for multiplexing. Each PCR reaction contained 11.5 μL post-RT cDNA library, 12.5 μL 2x KAPA HiFi HotStart PCR mix (Kapa Biosystems), and 1 μL of 25 μM PE1/PE2 index primer mix. Amplification was performed in 14 cycles. Amplified libraries were purified using 0.7x reaction volume of AMPure XP beads and eluted in 30 μL nuclease-free water. Aliquots of 15 μL of each resulting library were run in 2% agarose gels, and 200–700 bp DNA library fragments were isolated using PureLink DNA gel extraction kit (Invitrogen).  For all libraries quality was confirmed using an Agilent 2200 TapeStation nucleic acids system and an Agilent High Sensitivity D1000 DS DNA kit. Size-selected libraries were diluted to 4nM concentration and combined for paired-end, single index sequencing on the Illumina NextSeq 550 and 2000 instrument using an Illumina 550 High Output v2 (75 cycles) kit or NextSeq 1000/2000 P2 Reagents (100 Cycles) v3 kit. Cycle distribution was 45 cycles for Read 1, 35 cycles for Read 2, and 8 cycles for the library index read or 50 cycles for Read 1, 70 cycles for Read 2, and 8 cycles for the library indexes reads for Illumina 2000.