GSM3770930: A549 cells, sci-fate; Homo sapiens; RNA-Seq
All cell lines (A549, HEK293T and NIH/3T3 cells) were trypsinized, spun down at 300xg for 5 min (4°C) and washed once in 1X ice-cold PBS. All cells were fixed with 4ml ice cold 4% paraformaldehyde (EMS) for 15 min on ice. After fixation, cells were pelleted at 500xg for 3 min (4°C) and washed once with 1ml PBSR (1 x PBS, pH 7.4, 1% BSA, 1% SuperRnaseIn, 1% 10mM DTT). After wash, cells were resuspended in PBSR at 10 million cells per ml, and flash frozen and stored in liquid nitrogen. Paraformaldehyde fixed cells were thawed on 37°C water bath, spun down at 500xg for 5 min, and incubated with 500ul PBSR including 0.2% Triton X-100 for 3min on ice. Cells were pelleted and resuspended in 500ul nuclease free water including 1% SuperRnaseIn. 3ml 0.1N HCl were added into the cells for 5min incubation on ice. 3.5ml Tris-HCl (pH = 8.0) and 35ul 10% Triton X-100 were added into cells to neutralize HCl. Cells were pelleted and washed with 1ml PBSR. Cells were resuspended in 100ul PBSR. 100ul PBSR with fixed cells were incubated with mixture including 40ul Iodoacetamide (IAA, 100mM), 40ul sodium phosphate buffer (500mM, pH = 8.0), 200ul DMSO and 20ul H2O, at 50°C for 15min. The reaction was quenched by 8ul DTT (1M) and 8.5ml PBS. Cells were pelleted and resuspended in 100ul PBSI (1 x PBS, pH 7.4, 1% BSA, 1% SuperRnaseIn). For all later washes, nuclei were pelleted by centrifugation at 500xg for 5 min (4°C).   The following steps are similar with sci-RNA-seq protocol with paraformaldehyde fixed nuclei. Briefly, cells were distributed into four 96-well plates. For each well, 5,000 nuclei (2 µL) were mixed with 1 µl of 25 µM anchored oligo-dT primer (5′-ACGACGCTCTTCCGATCTNNNNNNNN[10bp index]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3′, where “N” is any base and “V” is either “A”, “C” or “G”; IDT) and 0.25 µL 10 mM dNTP mix (Thermo), denatured at 55°C for 5 min and immediately placed on ice. 1.75 µL of first-strand reaction mix, containing 1 µL 5X Superscript IV First-Strand Buffer (Invitrogen), 0.25 µl 100 mM DTT (Invitrogen), 0.25 µl SuperScript IV reverse transcriptase (200 U/μl, Invitrogen), 0.25 μL RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen), was then added to each well. Reverse transcription was carried out by incubating plates at the following temperature gradient: 4°C 2 minutes, 10°C 2 minutes, 20°C 2 minutes, 30°C 2 minutes, 40°C 2 minutes, 50°C 2 minutes and 55°C 10 minutes. All cells (or nuclei) were then pooled, stained with 4',6-diamidino-2-phenylindole (DAPI, Invitrogen) at a final concentration of 3 μM, and sorted at 25 nuclei per well into 5 μL EB buffer. Cells were gated based on DAPI stain such that singlets were discriminated from doublets and sorted into each well. 0.66 μl mRNA Second Strand Synthesis buffer (NEB) and 0.34 μl mRNA Second Strand Synthesis enzyme (NEB) were then added to each well, and second strand synthesis was carried out at 16°C for 180 min. Each well was then mixed with 5 μL Nextera TD buffer (Illumina) and 1 μL i7 only TDE1 enzyme (25 nM, Illumina, diluted in Nextera TD buffer), and then incubated at 55°C for 5 min to carry out tagmentation. The reaction was stopped by adding 12 μL DNA binding buffer (Zymo) and incubating at room temperature for 5 min. Each well was then purified using 36 uL AMPure XP beads (Beckman Coulter), eluted in 16 μL of buffer EB (Qiagen), then transferred to a fresh multi-well plate.  For PCR reactions, each well was mixed with 2μL of 10 μM P5 primer (5′-AATGATACGGCGACCACCGAGATCTACAC[i5]ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′; IDT), 2 μL of 10 μM P7 primer (5′-CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG-3′; IDT), and 20 μL NEBNext High-Fidelity 2X PCR Master Mix (NEB). Amplification was carried out using the following program: 72°C for 5 min, 98°C for 30 sec, 18-22 cycles of (98°C for 10 sec, 66°C for 30 sec, 72°C for 1 min) and a final 72°C for 5 min. After PCR, samples were pooled and purified using 0.8 volumes of AMPure XP beads. Library concentrations were determined by Qubit (Invitrogen) and the libraries were visualized by electrophoresis on a 6% TBE-PAGE gel. Libraries were sequenced on the NextSeq 500 platform (Illumina) using a V2 150 cycle kit (Read 1: 18 cycles, Read 2: 130 cycles, Index 1: 10 cycles, Index 2: 10 cycles).