GSM6340936: TABLE-seq_37C30min_rep2; Mus musculus; OTHER
RNA extraction was performed by RNeasy Plus Mini Kit (Qiagen, Cat. #74134). 2 μl 100 μM 5ph_Tn5a and 2 μl 100 μM Tn5a_N6_invert_dT were added to 16 μl H2O. Samples were annealed by 95 C for 5 min and ramped to 25 C at 5 C/min. 1 μl Exo I, 3 μl 10X Exo I reaction buffer, and 6 μl H2O were added and incubated at 37 C for 30 min, followed by 80 C for 15 min. The annealed adaptors were purified by 2X of VAHTS DNA clean beads, eluted into 20 μl H2O, and stored at -20 C until usage.    10 μl tagmented ssDNA or cDNA was mixed with 2 μl annealed adaptors, 0.5 μl T4 DNA ligase (Takara, Cat. #2011A), 2 μl 10X ligation buffer, 2.5 μl 40% PEG6000, 3 μl H2O, and incubated at 20 C for 1 hour. The ligated DNA was purified by 0.8X VAHTS DNA clean beads and eluted into 10 μl H2O. The purified DNA was amplified by primers with sequencing indexes. 1 μl 10 μM forward primer, 1 μl 10 μM reverse primer, 8 μl DNA, and 10 μl 2X HIFI PCR Master Mix (NEB, Cat. #M0541L) were mixed and amplified at 72 C for 5 min, 98 C for 30 sec, 20 cycles of 98 C for 10 sec and 63 C for 10 sec, then 72 C for 1 min, 16 C to hold. PCR products were purified by 0.9X VAHTS DNA clean beads and eluted into 20 μl H2O. The concentrations of DNA were detected by Equalbit 1X dsDNA HS Working Solution (Vazyme, Cat. #121-01-AA). The libraries were sequenced on an Illumina NovaSeq platform with pair-end reads of 150 bp. TABLE-seq