Generation of Stereo-seq chip.

Generation of Stereo-seq chip

To generate the patterned array, we first synthesized two oligo sequences: one containing 25 random deoxynucleotides ( TGTGAGCCAAGGAGTTGNAACTGCTGACGTACTGAGAGGCATGGCGACCTTATCAGNNNNNNNNNNNNNNNNNNNNNNNNNTTGTCTTCCTAAGACCG , Sangon) and the other a fixed sequence with phosphorylation ( /5phos/CTTGGCCTCCGACTTAAGTCGGATCGTAGCCATGTCGTTC, Sangon). These two oligos were ligated by incubation with T4 ligase (NEB; 1U/μl T4 DNA ligase and 1× T4 DNA ligation buffer) and splint oligo ( TCGGAGGCCAAGCGGTCTTAGGAA, Sangon, 1 μM) at 37℃ for 2 hours. The products were purified using the Ampure XP Beads (Vazyme, N411-03) and then PCR amplified with the following steps: 95℃ for 5 minutes, 12 cycles at 98℃ for 20 seconds, 58℃ for 20 seconds, 72℃ for 20 seconds and a final incubation at 72℃ for 5 minutes. The PCR products were purified using the Ampure XP Beads (Vazyme, N411-03). DNBs were then generated by rolling circle amplification and loaded onto the patterned chips according to the MGI DNBSEQ-Tx sequencer manual. Next, to determine the distinct DNB-CID sequences at each spatial location, single-end sequencing was performed using a sequencing primer ( CTGCTGACGTACTGAGAGGCATGGCGACCTTATCAG, Sangon, 1 μM) in a MGI DNBSEQ-Tx sequencer with SE25 sequencing strategy. After sequencing, the capture oligo ( /5phos/TTGTCTTCCTAAGACNNNNNNNNNNTTTTTTTTTTTTTTTTTTTTTV, Sangon, 1 μM) including 22 nt poly-T and 10 nt UMI was hybridized with the DNBs in 5× SSC buffer at 37℃ for 30 minutes, and then incubated with T4 ligase (NEB, 1 U/μl T4 DNA ligase, 1× T4 DNA ligation buffer and 0.5% PEG2000) at 37℃ for 1 hour. This procedure produces capture probes containing a 25 nt CID barcode, a 10 nt UMI and a 22 nt poly-T ready for poly-A RNA capture.