GSM4512670
GSM4512670: Neu-Kcl-15min; Mus musculus; RNA-Seq
After 4sU-labeling, K562 cells were pelleted and washed once with DPBS. mESCs were rinsed once with DPBS and dissociated with TrypLE-Express (Gibco, 12605010) for 5 min at 37°C. After neutralizing with culture medium, cells were pelleted at 1,000 rpm for 3 min. The cells were resuspended with DPBS-0.01% BSA. After cell counting with Countess II system, the single cell suspension was diluted to 100 cells/uL with DPBS containing 0.01% BSA and mixed at 1:1 ratio (mESCs:K562) for Drop-seq or scNT-seq analysis. Single cells, polyA-containing barcoded beads (ChemGenes), and droplet generation oil (Bio-Rad) were each flowed into individual input ports on a microfluidic device (uFluidix). Cells were lysed and hybridized to beads in droplets. Beads were isolated and subjected to reverse transcription (Thermo Fisher) and exonuclease I (NEB) treatment, followed by PCR of full length transcripts (Kapa). Final libraries were created by tagmentation of cDNA, amplified by PCR (Illumina, Nextera xT DNA Library Preparation kit) and purified by SPRI beads. For scNT-seq, the beads were treated with either TimeLapse chemistry (TFEA) or SLAM-seq chemistry (IAA) to convert 4sU to cytidine-analog before reverse transcription. For second-strand synthesis, after exonuclease I treatment, the beads were washed once with TE-SDS buffer and twice with TE-TW buffer. After aspiration of TE-TW wash, the beads were resuspended in 500 μL 0.1 M NaOH and incubated for 5 min at room temperature with rotation. Then 500 μL 0.2 M Tris-HCl (pH 7.5) was added to neutralize the solution. The beads were washed once with TE-TW buffer and once with 10 mM Tris-HCl (pH 8.0), then 200 μL 2nd-strand synthesis mix (1x Blue buffer (Enzymatics), 4% Ficoll PM-400, 1 mM dNTPs (Clontech), 2.5 μM Template Switch Oligo-GAATG (TSO-GAATG: /5SpC3/AAGCAGTGGTATCAACGCAGAGTGAATG), 5 μM TSO-N9 (TSO-N9: /5SpC3/AAGCAGTGGTATCAACGCAGAGTGAAT(N1:25252525)(N1) (N1)(N1)(N1)(N1)(N1)(N1)(N1)), and 1.25 U/ μL Klenow exo- (Enzymatics)) were added [N1 stands for mixture of A,T,C,G at 25:25:25:25 ratio]. The reaction was incubated at room temperature for 10 min, followed by incubation at 37°C for 1 hour with end-over-end rotation. The reaction was stopped by washing the beads once with TE-SDS buffer and twice with TE-TW buffer.
GEO
GSM4512670
GEO Accession
GSM4512670