Single cell intracellular capture method and application thereof
A cell, transfected cell technology, applied in biochemical equipment and methods, microbial determination/inspection, instruments, etc., can solve problems such as high cost of sequencing libraries
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Embodiment 1
[0118] Preparation of barcoded beads for poly-T extension clones
[0119] TELL beads, 3 μm clone barcoded beads from TELL-Seq WGS Library PrepKit (UST Corporation, PN #100000). The 3' end poly-T extended TELL beads are made with Pfu DNA polymerase and poly A-UMI (Unique Molecular Identifier) oligonucleotide A22-tUMI10
[0120] (5′-NBAAAAAAAAAAAAAAAAAAAAAABNNNNNNNNNNNGTGACCTGTCCCAGCGTCTCCAC-3′) prepared for primer extension of TELL beads as follows ( figure 1 ).
[0121] Eight 50 µL reactions were prepared including 1x Pfu buffer, 1 mM dNTPs, 2.5 mM MgCl2, 0.5 µM A22-tUMI10, 20 million TELL beads, and 0.06 U / µL Pfu polymerase. The following PCR program was used: 95°C for 1 minute, then 10 cycles of 95°C for 10 seconds, 62°C for 45 seconds, 72°C for 45 seconds, then 72°C for 3 minutes. After PCR, all beads were pooled and washed three times with bead wash buffer (10 mM Tris HCl, 0.1 mM EDTA, 0.1% Tween, pH 8). The beads will then be stripped by resuspending the beads in 50...
Embodiment 2
[0123] Transfection of barcoded beads into cells for intracellular capture
[0124] Add 10% FBS (Thermo Fisher Scientific, PN#26140-079), 1x Penicillin / Streptomycin (Thermo Fisher Scientific, PN#15140-122), 1xGlutamax (Thermo Fisher Scientific, PN#35050-061) and 0.05mM 2-mercaptoethanol (Thermo Fisher Scientific, PN#21985-023) in DMEM medium (Thermo Fisher Scientific, PN #11965-092) to culture and maintain HCT116 cells. For RNA extraction, when cells reached -75% confluency (approximately 1 million cells), cells were lysed and RNA was purified using Qiagen's RNeasy kit (Qiagen, PN #74104). Follow the manufacturer's protocol for on-column DNase treatment (Qiagen, PN #79254) and RNA purification steps (additional DNase treatment required). RNA was quantified using the BroadRange Qubit assay (Thermo Fisher Scientific, PN #Q10210).
[0125]Once the HCT116 cells reached approximately 80-90% confluency (approximately 1-1.5 M cells), cells were transfected with poly-T extended TEL...
Embodiment 3
[0127] In situ reverse transcription in bead-transfected living cells
[0128] The Superscript IV First-Strand Synthesis System kit (Superscript IV First-Strand Synthesis System kit) (Thermo Fisher Scientific, PN#18091050) was used to perform reverse transcription (RT) on living cells. Using approximately 150,000 bead-transfected cells from Example 2 as input, the manufacturer's recommended protocol was followed. 500,000 poly-T extended TELL beads with 500ng total RNA were used as a positive control and no reverse transcriptase as a negative control. Final RNase H treatment as described in the manufacturer's protocol was not performed. After reverse transcription, add 200 µL of resuspension buffer to the RT mix and purify by capturing beads / cells on a magnet for 2 min. The solution is removed and only the cells / beads are left attached to one side of the tube. A total of three washes were performed and the final beads / cells were resuspended in 25 μL of resuspension buffer. ...
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