Protocol Citation: Pengfei Guo, Yanxiang Deng 2024. spatial-Mux-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g71n79gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 25, 2024
Last Modified: October 22, 2024
Protocol Integer ID: 106407
Keywords: Spatial multi-omics, nano-CUT&Tag, co-profiling of five modalities
Abstract
Spatial-Mux-seq is a multi-modal spatial technology that allows simultaneous profiling of up to five different modalities, including multiple epigenomic modalities (any combination of two histone modifications and open chromatin at the same time), whole transcriptome, and a panel of proteins at tissue scale and cellular level in a spatially resolved manner. This was achieved by integrating microfluidic in situ barcoding and the nanobody-tethered transposition chemistry directly in tissue followed by high-throughput Next-Generation Sequencing.
Materials
Preparation of buffers:
DNA oligos used for PCR and preparation of sequencing library.
| RT-primer | /5Phos/CATCGGCGTACGACTNNNNNNNNNN/iBiodT/TTTTTTTTTTTTTTTVN | |
| template switch primer | AAGCAGTGGTATCAACGCAGAGTGAATrGrG+G | |
| Ligation linker 1 | AGTCGTACGCCGATGCGAAACATCGGCCAC | |
| Ligation linker 2 | CGAATGCTCTGGCCTCTCAAGCACGTGGAT | |
| PCR primer 1 | CAAGCGTTGGCTTCTCGCATCT | |
| PCR primer 2 | AAGCAGTGGTATCAACGCAGAGT | |
| primer 3 (cite-seq) | CCTTGGCACCCGAGAATT*C*C | |
| P5 oligo (cite-seq) | AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTTGGCACCCGAGAATTCC | |
| N501 | AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG | |
| N701 | CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT | |
| N702 | CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT | |
| N703 | CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT | |
| N704 | CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT | |
| N705 | CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT | |
| N706 | CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT | |
| N707 | CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT | |
| N709 | CAAGCAGAAGACGGCATACGAGATAGCGTAGCGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT | |
| N710 | CAAGCAGAAGACGGCATACGAGATCAGCCTCGGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT | |
| N711 | CAAGCAGAAGACGGCATACGAGATTGCCTCTTGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT | |
| N712 | CAAGCAGAAGACGGCATACGAGATTCCTCTACGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT |
Supplementary Table 5.
Barcode A Sequence
| Barcode A-1 | /5Phos/AGGCCAGAGCATTCGAACGTGATGTGGCCGATGTTTCG | |
| Barcode A-2 | /5Phos/AGGCCAGAGCATTCGAAACATCGGTGGCCGATGTTTCG | |
| Barcode A-3 | /5Phos/AGGCCAGAGCATTCGATGCCTAAGTGGCCGATGTTTCG | |
| Barcode A-4 | /5Phos/AGGCCAGAGCATTCGAGTGGTCAGTGGCCGATGTTTCG | |
| Barcode A-5 | /5Phos/AGGCCAGAGCATTCGACCACTGTGTGGCCGATGTTTCG | |
| Barcode A-6 | /5Phos/AGGCCAGAGCATTCGACATTGGCGTGGCCGATGTTTCG | |
| Barcode A-7 | /5Phos/AGGCCAGAGCATTCGCAGATCTGGTGGCCGATGTTTCG | |
| Barcode A-8 | /5Phos/AGGCCAGAGCATTCGCATCAAGTGTGGCCGATGTTTCG | |
| Barcode A-9 | /5Phos/AGGCCAGAGCATTCGCGCTGATCGTGGCCGATGTTTCG | |
| Barcode A-10 | /5Phos/AGGCCAGAGCATTCGACAAGCTAGTGGCCGATGTTTCG | |
| Barcode A-11 | /5Phos/AGGCCAGAGCATTCGCTGTAGCCGTGGCCGATGTTTCG | |
| Barcode A-12 | /5Phos/AGGCCAGAGCATTCGAGTACAAGGTGGCCGATGTTTCG | |
| Barcode A-13 | /5Phos/AGGCCAGAGCATTCGAACAACCAGTGGCCGATGTTTCG | |
| Barcode A-14 | /5Phos/AGGCCAGAGCATTCGAACCGAGAGTGGCCGATGTTTCG | |
| Barcode A-15 | /5Phos/AGGCCAGAGCATTCGAACGCTTAGTGGCCGATGTTTCG | |
| Barcode A-16 | /5Phos/AGGCCAGAGCATTCGAAGACGGAGTGGCCGATGTTTCG | |
| Barcode A-17 | /5Phos/AGGCCAGAGCATTCGAAGGTACAGTGGCCGATGTTTCG | |
| Barcode A-18 | /5Phos/AGGCCAGAGCATTCGACACAGAAGTGGCCGATGTTTCG | |
| Barcode A-19 | /5Phos/AGGCCAGAGCATTCGACAGCAGAGTGGCCGATGTTTCG | |
| Barcode A-20 | /5Phos/AGGCCAGAGCATTCGACCTCCAAGTGGCCGATGTTTCG | |
| Barcode A-21 | /5Phos/AGGCCAGAGCATTCGACGCTCGAGTGGCCGATGTTTCG | |
| Barcode A-22 | /5Phos/AGGCCAGAGCATTCGACGTATCAGTGGCCGATGTTTCG | |
| Barcode A-23 | /5Phos/AGGCCAGAGCATTCGACTATGCAGTGGCCGATGTTTCG | |
| Barcode A-24 | /5Phos/AGGCCAGAGCATTCGAGAGTCAAGTGGCCGATGTTTCG | |
| Barcode A-25 | /5Phos/AGGCCAGAGCATTCGAGATCGCAGTGGCCGATGTTTCG | |
| Barcode A-26 | /5Phos/AGGCCAGAGCATTCGAGCAGGAAGTGGCCGATGTTTCG | |
| Barcode A-27 | /5Phos/AGGCCAGAGCATTCGAGTCACTAGTGGCCGATGTTTCG | |
| Barcode A-28 | /5Phos/AGGCCAGAGCATTCGATCCTGTAGTGGCCGATGTTTCG | |
| Barcode A-29 | /5Phos/AGGCCAGAGCATTCGATTGAGGAGTGGCCGATGTTTCG | |
| Barcode A-30 | /5Phos/AGGCCAGAGCATTCGCAACCACAGTGGCCGATGTTTCG | |
| Barcode A-31 | /5Phos/AGGCCAGAGCATTCGGACTAGTAGTGGCCGATGTTTCG | |
| Barcode A-32 | /5Phos/AGGCCAGAGCATTCGCAATGGAAGTGGCCGATGTTTCG | |
| Barcode A-33 | /5Phos/AGGCCAGAGCATTCGCACTTCGAGTGGCCGATGTTTCG | |
| Barcode A-34 | /5Phos/AGGCCAGAGCATTCGCAGCGTTAGTGGCCGATGTTTCG | |
| Barcode A-35 | /5Phos/AGGCCAGAGCATTCGCATACCAAGTGGCCGATGTTTCG | |
| Barcode A-36 | /5Phos/AGGCCAGAGCATTCGCCAGTTCAGTGGCCGATGTTTCG | |
| Barcode A-37 | /5Phos/AGGCCAGAGCATTCGCCGAAGTAGTGGCCGATGTTTCG | |
| Barcode A-38 | /5Phos/AGGCCAGAGCATTCGCCGTGAGAGTGGCCGATGTTTCG | |
| Barcode A-39 | /5Phos/AGGCCAGAGCATTCGCCTCCTGAGTGGCCGATGTTTCG | |
| Barcode A-40 | /5Phos/AGGCCAGAGCATTCGCGAACTTAGTGGCCGATGTTTCG | |
| Barcode A-41 | /5Phos/AGGCCAGAGCATTCGCGACTGGAGTGGCCGATGTTTCG | |
| Barcode A-42 | /5Phos/AGGCCAGAGCATTCGCGCATACAGTGGCCGATGTTTCG | |
| Barcode A-43 | /5Phos/AGGCCAGAGCATTCGCTCAATGAGTGGCCGATGTTTCG | |
| Barcode A-44 | /5Phos/AGGCCAGAGCATTCGCTGAGCCAGTGGCCGATGTTTCG | |
| Barcode A-45 | /5Phos/AGGCCAGAGCATTCGCTGGCATAGTGGCCGATGTTTCG | |
| Barcode A-46 | /5Phos/AGGCCAGAGCATTCGGAATCTGAGTGGCCGATGTTTCG | |
| Barcode A-47 | /5Phos/AGGCCAGAGCATTCGCAAGACTAGTGGCCGATGTTTCG | |
| Barcode A-48 | /5Phos/AGGCCAGAGCATTCGGAGCTGAAGTGGCCGATGTTTCG | |
| Barcode A-49 | /5Phos/AGGCCAGAGCATTCGGATAGACAGTGGCCGATGTTTCG | |
| Barcode A-50 | /5Phos/AGGCCAGAGCATTCGGCCACATAGTGGCCGATGTTTCG | |
| Barcode A-51 | /5Phos/AGGCCAGAGCATTCGGCGAGTAAGTGGCCGATGTTTCG | |
| Barcode A-52 | /5Phos/AGGCCAGAGCATTCGGCTAACGAGTGGCCGATGTTTCG | |
| Barcode A-53 | /5Phos/AGGCCAGAGCATTCGGCTCGGTAGTGGCCGATGTTTCG | |
| Barcode A-54 | /5Phos/AGGCCAGAGCATTCGGGAGAACAGTGGCCGATGTTTCG | |
| Barcode A-55 | /5Phos/AGGCCAGAGCATTCGGGTGCGAAGTGGCCGATGTTTCG | |
| Barcode A-56 | /5Phos/AGGCCAGAGCATTCGGTACGCAAGTGGCCGATGTTTCG | |
| Barcode A-57 | /5Phos/AGGCCAGAGCATTCGGTCGTAGAGTGGCCGATGTTTCG | |
| Barcode A-58 | /5Phos/AGGCCAGAGCATTCGGTCTGTCAGTGGCCGATGTTTCG | |
| Barcode A-59 | /5Phos/AGGCCAGAGCATTCGGTGTTCTAGTGGCCGATGTTTCG | |
| Barcode A-60 | /5Phos/AGGCCAGAGCATTCGTAGGATGAGTGGCCGATGTTTCG | |
| Barcode A-61 | /5Phos/AGGCCAGAGCATTCGTATCAGCAGTGGCCGATGTTTCG | |
| Barcode A-62 | /5Phos/AGGCCAGAGCATTCGTCCGTCTAGTGGCCGATGTTTCG | |
| Barcode A-63 | /5Phos/AGGCCAGAGCATTCGTCTTCACAGTGGCCGATGTTTCG | |
| Barcode A-64 | /5Phos/AGGCCAGAGCATTCGTGAAGAGAGTGGCCGATGTTTCG | |
| Barcode A-65 | /5Phos/AGGCCAGAGCATTCGTGGAACAAGTGGCCGATGTTTCG | |
| Barcode A-66 | /5Phos/AGGCCAGAGCATTCGTGGCTTCAGTGGCCGATGTTTCG | |
| Barcode A-67 | /5Phos/AGGCCAGAGCATTCGTGGTGGTAGTGGCCGATGTTTCG | |
| Barcode A-68 | /5Phos/AGGCCAGAGCATTCGTTCACGCAGTGGCCGATGTTTCG | |
| Barcode A-69 | /5Phos/AGGCCAGAGCATTCGAACTCACCGTGGCCGATGTTTCG | |
| Barcode A-70 | /5Phos/AGGCCAGAGCATTCGAAGAGATCGTGGCCGATGTTTCG | |
| Barcode A-71 | /5Phos/AGGCCAGAGCATTCGAAGGACACGTGGCCGATGTTTCG | |
| Barcode A-72 | /5Phos/AGGCCAGAGCATTCGAATCCGTCGTGGCCGATGTTTCG | |
| Barcode A-73 | /5Phos/AGGCCAGAGCATTCGAATGTTGCGTGGCCGATGTTTCG | |
| Barcode A-74 | /5Phos/AGGCCAGAGCATTCGACACGACCGTGGCCGATGTTTCG | |
| Barcode A-75 | /5Phos/AGGCCAGAGCATTCGACAGATTCGTGGCCGATGTTTCG | |
| Barcode A-76 | /5Phos/AGGCCAGAGCATTCGAGATGTACGTGGCCGATGTTTCG | |
| Barcode A-77 | /5Phos/AGGCCAGAGCATTCGAGCACCTCGTGGCCGATGTTTCG | |
| Barcode A-78 | /5Phos/AGGCCAGAGCATTCGAGCCATGCGTGGCCGATGTTTCG | |
| Barcode A-79 | /5Phos/AGGCCAGAGCATTCGAGGCTAACGTGGCCGATGTTTCG | |
| Barcode A-80 | /5Phos/AGGCCAGAGCATTCGATAGCGACGTGGCCGATGTTTCG | |
| Barcode A-81 | /5Phos/AGGCCAGAGCATTCGATCATTCCGTGGCCGATGTTTCG | |
| Barcode A-82 | /5Phos/AGGCCAGAGCATTCGATTGGCTCGTGGCCGATGTTTCG | |
| Barcode A-83 | /5Phos/AGGCCAGAGCATTCGCAAGGAGCGTGGCCGATGTTTCG | |
| Barcode A-84 | /5Phos/AGGCCAGAGCATTCGCACCTTACGTGGCCGATGTTTCG | |
| Barcode A-85 | /5Phos/AGGCCAGAGCATTCGCCATCCTCGTGGCCGATGTTTCG | |
| Barcode A-86 | /5Phos/AGGCCAGAGCATTCGCCGACAACGTGGCCGATGTTTCG | |
| Barcode A-87 | /5Phos/AGGCCAGAGCATTCGCCTAATCCGTGGCCGATGTTTCG | |
| Barcode A-88 | /5Phos/AGGCCAGAGCATTCGCCTCTATCGTGGCCGATGTTTCG | |
| Barcode A-89 | /5Phos/AGGCCAGAGCATTCGCGACACACGTGGCCGATGTTTCG | |
| Barcode A-90 | /5Phos/AGGCCAGAGCATTCGCGGATTGCGTGGCCGATGTTTCG | |
| Barcode A-91 | /5Phos/AGGCCAGAGCATTCGCTAAGGTCGTGGCCGATGTTTCG | |
| Barcode A-92 | /5Phos/AGGCCAGAGCATTCGGAACAGGCGTGGCCGATGTTTCG | |
| Barcode A-93 | /5Phos/AGGCCAGAGCATTCGGACAGTGCGTGGCCGATGTTTCG | |
| Barcode A-94 | /5Phos/AGGCCAGAGCATTCGGAGTTAGCGTGGCCGATGTTTCG | |
| Barcode A-95 | /5Phos/AGGCCAGAGCATTCGGATGAATCGTGGCCGATGTTTCG | |
| Barcode A-96 | /5Phos/AGGCCAGAGCATTCGGCCAAGACGTGGCCGATGTTTCG | |
| Barcode A-97 | /5Phos/AGGCCAGAGCATTCGCGGAAGAAGTGGCCGATGTTTCG | |
| Barcode A-98 | /5Phos/AGGCCAGAGCATTCGGTGACAAGGTGGCCGATGTTTCG | |
| Barcode A-99 | /5Phos/AGGCCAGAGCATTCGGAACCAGAGTGGCCGATGTTTCG | |
| Barcode A-100 | /5Phos/AGGCCAGAGCATTCGTTGCTGGAGTGGCCGATGTTTCG |
Supplementary Table 6.
Barcode B Sequence
| Barcode B-1 | AAGCGTTGGCTTCTCGCATCTAACGTGATATCCACGTGCTTGAG | |
| Barcode B-2 | CAAGCGTTGGCTTCTCGCATCTAAACATCGATCCACGTGCTTGAG | |
| Barcode B-3 | CAAGCGTTGGCTTCTCGCATCTATGCCTAAATCCACGTGCTTGAG | |
| Barcode B-4 | CAAGCGTTGGCTTCTCGCATCTAGTGGTCAATCCACGTGCTTGAG | |
| Barcode B-5 | CAAGCGTTGGCTTCTCGCATCTACCACTGTATCCACGTGCTTGAG | |
| Barcode B-6 | CAAGCGTTGGCTTCTCGCATCTACATTGGCATCCACGTGCTTGAG | |
| Barcode B-7 | CAAGCGTTGGCTTCTCGCATCTCAGATCTGATCCACGTGCTTGAG | |
| Barcode B-8 | CAAGCGTTGGCTTCTCGCATCTCATCAAGTATCCACGTGCTTGAG | |
| Barcode B-9 | CAAGCGTTGGCTTCTCGCATCTCGCTGATCATCCACGTGCTTGAG | |
| Barcode B-10 | CAAGCGTTGGCTTCTCGCATCTACAAGCTAATCCACGTGCTTGAG | |
| Barcode B-11 | CAAGCGTTGGCTTCTCGCATCTCTGTAGCCATCCACGTGCTTGAG | |
| Barcode B-12 | CAAGCGTTGGCTTCTCGCATCTAGTACAAGATCCACGTGCTTGAG | |
| Barcode B-13 | CAAGCGTTGGCTTCTCGCATCTAACAACCAATCCACGTGCTTGAG | |
| Barcode B-14 | CAAGCGTTGGCTTCTCGCATCTAACCGAGAATCCACGTGCTTGAG | |
| Barcode B-15 | CAAGCGTTGGCTTCTCGCATCTAACGCTTAATCCACGTGCTTGAG | |
| Barcode B-16 | CAAGCGTTGGCTTCTCGCATCTAAGACGGAATCCACGTGCTTGAG | |
| Barcode B-17 | CAAGCGTTGGCTTCTCGCATCTAAGGTACAATCCACGTGCTTGAG | |
| Barcode B-18 | CAAGCGTTGGCTTCTCGCATCTACACAGAAATCCACGTGCTTGAG | |
| Barcode B-19 | CAAGCGTTGGCTTCTCGCATCTACAGCAGAATCCACGTGCTTGAG | |
| Barcode B-20 | CAAGCGTTGGCTTCTCGCATCTACCTCCAAATCCACGTGCTTGAG | |
| Barcode B-21 | CAAGCGTTGGCTTCTCGCATCTACGCTCGAATCCACGTGCTTGAG | |
| Barcode B-22 | CAAGCGTTGGCTTCTCGCATCTACGTATCAATCCACGTGCTTGAG | |
| Barcode B-23 | CAAGCGTTGGCTTCTCGCATCTACTATGCAATCCACGTGCTTGAG | |
| Barcode B-24 | CAAGCGTTGGCTTCTCGCATCTAGAGTCAAATCCACGTGCTTGAG | |
| Barcode B-25 | CAAGCGTTGGCTTCTCGCATCTAGATCGCAATCCACGTGCTTGAG | |
| Barcode B-26 | CAAGCGTTGGCTTCTCGCATCTAGCAGGAAATCCACGTGCTTGAG | |
| Barcode B-27 | CAAGCGTTGGCTTCTCGCATCTAGTCACTAATCCACGTGCTTGAG | |
| Barcode B-28 | CAAGCGTTGGCTTCTCGCATCTATCCTGTAATCCACGTGCTTGAG | |
| Barcode B-29 | CAAGCGTTGGCTTCTCGCATCTATTGAGGAATCCACGTGCTTGAG | |
| Barcode B-30 | CAAGCGTTGGCTTCTCGCATCTCAACCACAATCCACGTGCTTGAG | |
| Barcode B-31 | CAAGCGTTGGCTTCTCGCATCTGACTAGTAATCCACGTGCTTGAG | |
| Barcode B-32 | CAAGCGTTGGCTTCTCGCATCTCAATGGAAATCCACGTGCTTGAG | |
| Barcode B-33 | CAAGCGTTGGCTTCTCGCATCTCACTTCGAATCCACGTGCTTGAG | |
| Barcode B-34 | CAAGCGTTGGCTTCTCGCATCTCAGCGTTAATCCACGTGCTTGAG | |
| Barcode B-35 | CAAGCGTTGGCTTCTCGCATCTCATACCAAATCCACGTGCTTGAG | |
| Barcode B-36 | CAAGCGTTGGCTTCTCGCATCTCCAGTTCAATCCACGTGCTTGAG | |
| Barcode B-37 | CAAGCGTTGGCTTCTCGCATCTCCGAAGTAATCCACGTGCTTGAG | |
| Barcode B-38 | CAAGCGTTGGCTTCTCGCATCTCCGTGAGAATCCACGTGCTTGAG | |
| Barcode B-39 | CAAGCGTTGGCTTCTCGCATCTCCTCCTGAATCCACGTGCTTGAG | |
| Barcode B-40 | CAAGCGTTGGCTTCTCGCATCTCGAACTTAATCCACGTGCTTGAG | |
| Barcode B-41 | CAAGCGTTGGCTTCTCGCATCTCGACTGGAATCCACGTGCTTGAG | |
| Barcode B-42 | CAAGCGTTGGCTTCTCGCATCTCGCATACAATCCACGTGCTTGAG | |
| Barcode B-43 | CAAGCGTTGGCTTCTCGCATCTCTCAATGAATCCACGTGCTTGAG | |
| Barcode B-44 | CAAGCGTTGGCTTCTCGCATCTCTGAGCCAATCCACGTGCTTGAG | |
| Barcode B-45 | CAAGCGTTGGCTTCTCGCATCTCTGGCATAATCCACGTGCTTGAG | |
| Barcode B-46 | CAAGCGTTGGCTTCTCGCATCTGAATCTGAATCCACGTGCTTGAG | |
| Barcode B-47 | CAAGCGTTGGCTTCTCGCATCTCAAGACTAATCCACGTGCTTGAG | |
| Barcode B-48 | CAAGCGTTGGCTTCTCGCATCTGAGCTGAAATCCACGTGCTTGAG | |
| Barcode B-49 | CAAGCGTTGGCTTCTCGCATCTGATAGACAATCCACGTGCTTGAG | |
| Barcode B-50 | CAAGCGTTGGCTTCTCGCATCTGCCACATAATCCACGTGCTTGAG | |
| Barcode B-51 | CAAGCGTTGGCTTCTCGCATCTGCGAGTAAATCCACGTGCTTGAG | |
| Barcode B-52 | CAAGCGTTGGCTTCTCGCATCTGCTAACGAATCCACGTGCTTGAG | |
| Barcode B-53 | CAAGCGTTGGCTTCTCGCATCTGCTCGGTAATCCACGTGCTTGAG | |
| Barcode B-54 | CAAGCGTTGGCTTCTCGCATCTGGAGAACAATCCACGTGCTTGAG | |
| Barcode B-55 | CAAGCGTTGGCTTCTCGCATCTGGTGCGAAATCCACGTGCTTGAG | |
| Barcode B-56 | CAAGCGTTGGCTTCTCGCATCTGTACGCAAATCCACGTGCTTGAG | |
| Barcode B-57 | CAAGCGTTGGCTTCTCGCATCTGTCGTAGAATCCACGTGCTTGAG | |
| Barcode B-58 | CAAGCGTTGGCTTCTCGCATCTGTCTGTCAATCCACGTGCTTGAG | |
| Barcode B-59 | CAAGCGTTGGCTTCTCGCATCTGTGTTCTAATCCACGTGCTTGAG | |
| Barcode B-60 | CAAGCGTTGGCTTCTCGCATCTTAGGATGAATCCACGTGCTTGAG | |
| Barcode B-61 | CAAGCGTTGGCTTCTCGCATCTTATCAGCAATCCACGTGCTTGAG | |
| Barcode B-62 | CAAGCGTTGGCTTCTCGCATCTTCCGTCTAATCCACGTGCTTGAG | |
| Barcode B-63 | CAAGCGTTGGCTTCTCGCATCTTCTTCACAATCCACGTGCTTGAG | |
| Barcode B-64 | CAAGCGTTGGCTTCTCGCATCTTGAAGAGAATCCACGTGCTTGAG | |
| Barcode B-65 | CAAGCGTTGGCTTCTCGCATCTTGGAACAAATCCACGTGCTTGAG | |
| Barcode B-66 | CAAGCGTTGGCTTCTCGCATCTTGGCTTCAATCCACGTGCTTGAG | |
| Barcode B-67 | CAAGCGTTGGCTTCTCGCATCTTGGTGGTAATCCACGTGCTTGAG | |
| Barcode B-68 | CAAGCGTTGGCTTCTCGCATCTTTCACGCAATCCACGTGCTTGAG | |
| Barcode B-69 | CAAGCGTTGGCTTCTCGCATCTAACTCACCATCCACGTGCTTGAG | |
| Barcode B-70 | CAAGCGTTGGCTTCTCGCATCTAAGAGATCATCCACGTGCTTGAG | |
| Barcode B-71 | CAAGCGTTGGCTTCTCGCATCTAAGGACACATCCACGTGCTTGAG | |
| Barcode B-72 | CAAGCGTTGGCTTCTCGCATCTAATCCGTCATCCACGTGCTTGAG | |
| Barcode B-73 | CAAGCGTTGGCTTCTCGCATCTAATGTTGCATCCACGTGCTTGAG | |
| Barcode B-74 | CAAGCGTTGGCTTCTCGCATCTACACGACCATCCACGTGCTTGAG | |
| Barcode B-75 | CAAGCGTTGGCTTCTCGCATCTACAGATTCATCCACGTGCTTGAG | |
| Barcode B-76 | CAAGCGTTGGCTTCTCGCATCTAGATGTACATCCACGTGCTTGAG | |
| Barcode B-77 | CAAGCGTTGGCTTCTCGCATCTAGCACCTCATCCACGTGCTTGAG | |
| Barcode B-78 | CAAGCGTTGGCTTCTCGCATCTAGCCATGCATCCACGTGCTTGAG | |
| Barcode B-79 | CAAGCGTTGGCTTCTCGCATCTAGGCTAACATCCACGTGCTTGAG | |
| Barcode B-80 | CAAGCGTTGGCTTCTCGCATCTATAGCGACATCCACGTGCTTGAG | |
| Barcode B-81 | CAAGCGTTGGCTTCTCGCATCTATCATTCCATCCACGTGCTTGAG | |
| Barcode B-82 | CAAGCGTTGGCTTCTCGCATCTATTGGCTCATCCACGTGCTTGAG | |
| Barcode B-83 | CAAGCGTTGGCTTCTCGCATCTCAAGGAGCATCCACGTGCTTGAG | |
| Barcode B-84 | CAAGCGTTGGCTTCTCGCATCTCACCTTACATCCACGTGCTTGAG | |
| Barcode B-85 | CAAGCGTTGGCTTCTCGCATCTCCATCCTCATCCACGTGCTTGAG | |
| Barcode B-86 | CAAGCGTTGGCTTCTCGCATCTCCGACAACATCCACGTGCTTGAG | |
| Barcode B-87 | CAAGCGTTGGCTTCTCGCATCTCCTAATCCATCCACGTGCTTGAG | |
| Barcode B-88 | CAAGCGTTGGCTTCTCGCATCTCCTCTATCATCCACGTGCTTGAG | |
| Barcode B-89 | CAAGCGTTGGCTTCTCGCATCTCGACACACATCCACGTGCTTGAG | |
| Barcode B-90 | CAAGCGTTGGCTTCTCGCATCTCGGATTGCATCCACGTGCTTGAG | |
| Barcode B-91 | CAAGCGTTGGCTTCTCGCATCTCTAAGGTCATCCACGTGCTTGAG | |
| Barcode B-92 | CAAGCGTTGGCTTCTCGCATCTGAACAGGCATCCACGTGCTTGAG | |
| Barcode B-93 | CAAGCGTTGGCTTCTCGCATCTGACAGTGCATCCACGTGCTTGAG | |
| Barcode B-94 | CAAGCGTTGGCTTCTCGCATCTGAGTTAGCATCCACGTGCTTGAG | |
| Barcode B-95 | CAAGCGTTGGCTTCTCGCATCTGATGAATCATCCACGTGCTTGAG | |
| Barcode B-96 | CAAGCGTTGGCTTCTCGCATCTGCCAAGACATCCACGTGCTTGAG | |
| Barcode B-97 | CAAGCGTTGGCTTCTCGCATCTCGGAAGAAATCCACGTGCTTGAG | |
| Barcode B-98 | CAAGCGTTGGCTTCTCGCATCTGTGACAAGATCCACGTGCTTGAG | |
| Barcode B-99 | CAAGCGTTGGCTTCTCGCATCTGAACCAGAATCCACGTGCTTGAG | |
| Barcode B-100 | CAAGCGTTGGCTTCTCGCATCTTTGCTGGAATCCACGTGCTTGAG |
Supplementary Table 7.
Chemicals and reagents
| A | B | C | |
| Name | Catalog number | Vender | |
| Formaldehyde solution | PI28906 | Thermo Fisher Scientific | |
| HEPES pH 7.5 | BBH-75-250 | Boston BioProducts | |
| Glycine | 50046 | Sigma-Aldrich | |
| NaCl | AM9760G | Thermo Fisher Scientific | |
| Digitonin | G9441 | Promega | |
| MgCl2 | AM9530G | Thermo Fisher Scientific | |
| Spermidine | S0266 | Sigma-Aldrich | |
| EDTA-free Protease Inhibitor Cocktail | 11873580001 | Millipore Sigma | |
| NP40 | 11332473001 | Sigma-Aldrich | |
| EDTA Solution pH 8.0 | AB00502 | AmericanBio | |
| Bovine Serum Albumin (BSA) | A8806 | Sigma-Aldrich | |
| Anti-H3K27me3 antibody | Ab6002 | Abcam | |
| Anti-H3K27ac antibody | 8173 | Cell Signaling Technology | |
| Anti-H3K4me3 antibody | 9751 | Cell Signaling Technology | |
| TotalSeq‱-A Mouse Universal Cocktail | 199901 | Biolegend | |
| Anti mouse CD4 | A0001 | Biolegend | |
| Anti mouse CD3 | A0182 | Biolegend | |
| Anti mouse CD34 | A0857 | Biolegend | |
| Anti mouse CD140a | A0573 | Biolegend | |
| Anti mouse CD133 | A1037 | Biolegend | |
| Anti mouse CD90.1 | A0380 | Biolegend | |
| Anti mouse CD90.2 | A0075 | Biolegend | |
| Anti mouse B220 | A0103 | Biolegend | |
| TruStain FcX‱ PLUS (anti-mouse CD16/32) antibody | 156604 | Biolegend | |
| Cell Staining Buffer | 420201 | Biolegend | |
| Fab Fragment Goat Anti-Mouse IgG | 115-007-003 | Jackson ImmunoResearch | |
| Triton X-100 | T8787 | Sigma-Aldrich | |
| T4 DNA Ligase | M0202L | New England Biolabs | |
| T4 DNA Ligase Reaction Buffer | B0202S | New England Biolabs | |
| NEBuffer 3.1 | B7203S | New England Biolabs | |
| DPBS | 14190144 | Thermo Fisher Scientific | |
| Proteinase K | EO0491 | Thermo Fisher Scientific | |
| SPRI beads | A63880 | Beckman Coulter | |
| NEBNext High-Fidelity 2X PCR Master Mix | M0541L | New England Biolabs | |
| SYBR Green I Nucleic Acid Gel Stain | S7563 | Thermo Fisher Scientific | |
| DNA Clean & Concentrator-5 | D4014 | Zymo Research | |
| Tn5 Transposase - unloaded | C01070010 | Diagenode | |
| Tagmentation Buffer (2x) | C01019043 | Diagenode | |
| Sodium dodecyl sulfate | 71736 | Sigma-Aldrich | |
| Maxima H Minus Reverse Transcriptase (200 U/L) | EP0751 | Thermo Fisher Scientific | |
| dNTP mix | R0192 | Thermo Fisher Scientific | |
| Dynabeads‱ MyOne‱ Streptavidin C1 | 65001 | Thermo Fisher Scientific | |
| RNase Inhibitor | Y9240L | Enzymatics | |
| Kapa Hotstart HiFi ReadyMix | KK2601 | Kapa Biosystems | |
| Nextera XT DNA Library Preparation Kit | FC-131-1024 | Illumina |
Safety warnings
Digitonin is toxic and care should be taken especially when weighing out the powder. Use full PPE including a mask, lab coat and gloves while handling any amount of digitonin.
Cryosectioning Procedure
Cryosectioning Procedure
Specimens should be handled with RNAse Away.
Note
This protocol is to be used with tissue preserved in OCT and stored in -80 °C .
- Cool Cryostat to -20 °C . Clean the work surfaces with RNAse away and change a new cutting blade.
- Place a clean slide box inside the cryostat chamber to store slides.
- Clean the tissue holder with RNAse away and put it in the cryostat chamber . Adhere the OCT specimen to a tissue holder and allow it to equilibrate for 00:20:00 to reach the chamber temperature and strengthen the adhesion between the OCT block and the holder.
- Place the specimen in the cryostat with the tissue holder and cut at Thikness 8-10 µm.
- Store completed slides at -80 °C .
20m
Tn5 loading
Tn5 loading
10m
10m
Annealing adaptor sequences:
Tn5MErev:
5′-/Phos/CTGTCTCTTATACACATCT-3′
Tn5ME-A:
5′-/Phos/CATCGGCGTACGACTAGATGTGTATAAGAGACAG-3′
Tn5ME-B1:
5′-/Phos/CATCGGCGTACGACTTAGCCTAGATGTGTATAAGAGACAG-3′
Tn5ME-B2:
5’-/Phos/CATCGGCGTACGACTATAGAGAGATGTGTATAAGAGACAG-3′
Tn5ME-B3:
5’-/Phos/CATCGGCGTACGACTCCTATCAGATGTGTATAAGAGACAG-3′
- Resuspend the oligonucleotides (Tn5MErev, Tn5ME-A, Tn5ME-B) in water to a final concentration of 100 µM each.
- Mix equimolar amounts of Tn5MErev/Tn5ME-A and Tn5MErev/Tn5ME-B in separate 200 µl PCR tubes.
e.g:
tube1: 10 µL Tn5ME-A + 10 µL Tn5MErev
tube2: 10 µL Tn5ME-B + 10 µL Tn5MErev
- Denature in the thermocycler for 00:05:00 at 95 °C , and cool down slowly on the thermocycler by ramping down to 12 °C by 0.1 °C per second
Pause point: Store the annealed oligos at -20 °C
- Mix annealing mix.
The final Tn5 concentration is 2 µM of Tn5 dimer.
Use unique barcodes for specific nano-Tn5 or WT-Tn5.
2x Tn5 loading buffer
Mouse Nano-Tn5 (MeA/B1/R loaded):
4 µL Annealed, barcoded MeA/Me-Rev oligos Concentration50 micromolar (µM)
4 µL Annealed, barcoded MeB1/Me-Rev oligos Concentration50 micromolar (µM)
42 µL Glycerol
6 µL Nano-Tn5 (mouse, 5mg/ml, MW = 73941 g/mol; Concentration67.6 micromolar (µM) )
44 µL 2x Nano-Tn5 loading buffer
Rabbit Nano-Tn5 (MeA/B2/R loaded):
4 µL Annealed, barcoded MeA/Me-Rev oligos Concentration50 micromolar (µM)
4 µL Annealed, barcoded MeB2/Me-Rev oligos Concentration50 micromolar (µM)
42 µL Glycerol
4.4 µL Nano-Tn5 (rabbit, 6.8mg/ml, MW = 73013 g/mol Concentration93.1 micromolar (µM) )
45.6 µL 2x Nano-Tn5 loading buffer
For nano-Tn5, incubate for 01:00:00 at 23 °C .
WT Tn5-MeA with barcode (MeA/B3/R for ATAC-seq)
5 µL Annealed, barcoded MeA/Me-Rev oligos Concentration50 micromolar (µM)
5 µL Annealed, barcoded MeB3/Me-Rev oligos Concentration50 micromolar (µM)
10 µL Tn5 (Order from Diagenode)
For WT-Tn5, incubate for 00:30:00 at 23 °C .
Then mix with 10 µL Glycerol.
Note
Store the loaded nano-Tn5 or WT-Tn5 at Temperature-20 °C.
1h 35m
Fixation
Fixation
Frozen tissue slides were first thawed for 00:01:00 at 37 °C .
1m
Tissue was fixed with 1 mL 0.2 % volume formaldehyde in PBS (with 0.05 U μl–1 RNase Inhibitor) at Room temperature for 00:05:00 .
5m
Discard reagent and quench with 1 mL 1.25 Molarity (M) glycine (with 0.05 U μl–1 RNase Inhibitor) for 00:05:00 .
5m
Discard reagent and wash slide with 1x PBS (with 0.05 U μl–1 RNase Inhibitor) for 00:01:00 .
1m
Discard reagent and rinse slide sequentially in RNase free water. Wipe away excess liquid.
Allow slide to air dry and image the slide on a Keyence BZ-X810 scanning microscope at 10x or 20x resolution.
ATAC-seq
ATAC-seq
10m
10m
Buffer preparation for ATAC-seq
Attach the PDMS reservoir and 12mm diameter clamp to the tissue.
Note
The PDMS reservoir should cover the entire tissue region of interest on the slide, but small enough to save reagents.
Permeabilize the tissue with 200 µL 0.1X lysis buffer and incubate at Room temperature for00:15:00 .
15m
Discard reagent and Wash the tissue with 200 µL wash buffer I at Room temperature for00:05:00 .
5m
Discard reagent and quickly wash the tissue with 200 µL wash buffer.
Prepare transposition mix:
50 µL 2X TD Buffer (see materials section)
33 µL 1X PBS
1 µL 10% Tween-20 (final 0.1% v/v)
1 µL 1% Digitonin (final 0.01% v/v)
5 µL Tn5 Transposase (WT Tn5)
10 µL nuclease-free H2O
Discard reagent and add 100 µL transposition mix.
Incubate for 00:30:00 at 37 °C .
30m
Discard reagent and stop the tagmentation by adding 200 µL of 40 millimolar (mM) EDTA atRoom temperature for 00:05:00 .
5m
Nano-CUT&Tag
Nano-CUT&Tag
Buffer preparation for Nano-CUT&Tag
Discard reagent and Wash the tissue with 200 µL wash buffer II at Room temperature for00:05:00 .
5m
Discard reagent and Wash the tissue with 200 µL NP40-Digitonin wash buffer at Room temperature for00:05:00 .
5m
Primary antibody binding
Primary antibody binding
Starting concentrations (can be further optimised, depending on the antibody)
1:50 primary antibody in antibody-binding buffer.
Final volume100 µL per sample.
Discard reagent and add 100 µL primary antibody mix.
Incubate at4 °C Overnight
Note
Make sure the liquid can cover the tissue region of interest.
20m
Nano-Tn5 binding
Nano-Tn5 binding
Discard reagent and Wash the tissue with 200 µL 300-wash buffer at Room temperature for00:05:00 .
5m
Starting concentrations of nano-Tn5s mix
1:100 nano-Tn5s in 300-wash buffer.
Final volume100 µL per sample.
Discard reagent and add 100 µL nano-Tn5s mix at Room temperature for01:00:00
1h
Discard reagent and Wash the tissue with 200 µL 300-wash buffer at Room temperature for00:05:00 .
5m
Discard reagent and add 200 µL Tagmentation buffer.
Incubate for 01:00:00 at 37 °C .
1h
Discard reagent and stop the tagmentation by adding 200 µL of 40 millimolar (mM) EDTA atRoom temperature for 00:05:00 .
5m
Surface Protein Antibody staining
Surface Protein Antibody staining
Discard reagent and add 200 µL of cell surface staining buffer at Room temperature for 00:02:00 .
2m
Discard reagent and add 200 µL of 1:20 TruStain FcX‱ in Cell Staining Buffer at4 °C for 00:15:00 .
15m
Discard reagent and add 200 µL of cell surface staining buffer at Room temperature for 00:01:00 .
1m
Discard reagent and add 200 µL of oligonucleotide-labeled antibody-derived tags (1:100) in cell surface staining buffer.
Incubate for 00:30:00 at 4 °C .
30m
Discard reagent and add 200 µL of cell surface staining buffer at Room temperature for 00:01:00 .
1m
Discard reagent and add 200 µL of Fab Fragment Goat Anti-Mouse IgG (1:25) in cell surface staining buffer.
Incubate for 00:15:00 at 4 °C .
15m
In situ reverse transcription
In situ reverse transcription
Dsicard reagent and tissue was fixed with 200 µL 2 % volume formaldehyde in PBS (with 0.05 U μl–1 RNase Inhibitor) at Room temperature for 00:10:00 .
10m
Discard reagent and quench with 1 mL 1.25 Molarity (M) glycine (with 0.05 U μl–1 RNase Inhibitor) for 00:05:00 .
5m
Discard reagent and add 200 µL of 0.5x PBS at Room temperature for 00:05:00 .
5m
Prepare the RT mixture
For one sample:
12.5 µL 5X RT Buffer
0.8 µL RNase inhibitor
3.1 µL dNTPs
6.1 µL Maxima H Minus Reverse Transcriptase
20.3 µL 0.5X PBS
10 µL RT primer
9.6 µL ddH2O
Discard reagent and add 62.5 µL RT mixture.
Incubate atRoom temperature 00:30:00 .
30m
Incubate at42 °C 01:30:00 .
1h 30m
Discard reagent and add 200 µL of 1X NEBBuffer 3.1 at Room temperature for 00:05:00 .
5m
Discard reagent, remove the clamp, and dip the tissue slide into ddH2O.
Air dry the slide.
In-tissue ligation with barcode A
In-tissue ligation with barcode A
Prepare buffer:
2X annealing buffer:
2 millimolar (mM) EDTA, pH 8.0
20 millimolar (mM) Tris-HCl, pH 7.5
100 millimolar (mM) NaCl
Prepare annealed barcode An
25 µL 2X annealing buffer
12.5 µL 100 micromolar (µM) barcode An
12.5 µL 100 micromolar (µM) ligation linker 1
Prepare annealed barcode Bn
25 µL 2X annealing buffer
12.5 µL 100 micromolar (µM) barcode Bn
12.5 µL 100 micromolar (µM) ligation linker 2
Run the following program:
95 °C 00:05:00 , and cool to 12 °C , -0.1 ℃/sec.
Note
Annealed barcodes can be stored at -20 °C
Prepare spatial barcode mix, for each channel
27 µL 10X T4 DNA ligase buffer
11 µL T4 DNA ligase
5.4 µL 5% Triton X-100
11.58 µL 10X NEB buffer3.1
175 µL Nuclease free water
Prepare spatial barcode mix, for each channel:
4 µL ligation mix
1 µL annealed barcode An (such as A1)
5m
Apply the microfluidic chip A to the tissue slide to cover the region of interest.
Note
Ensure the microfluidic chip is dust free by applying tape on the microfluid chip and tissue
slide to gently remove dust.
Image full chip A on the tissue slide with no clamp using Keyence microscopy with an 10× objective lens.
Load the slide into a clamp. Secure the clamp onto the slide, and tighten the screws.
Load the spatial barcode mix A to the inlets, and attach a vacuum gasket over the outlets.
Flow the chip until the solution has reached the outlet wells for all channels.
Remove the vacuum gasket and incubate the tissue slide at 37 °C for 00:30:00 .
30m
Suck the solution out of the inlet and outlet.
Dip the slide in RNase free water and air dry the slide.
In-tissue ligation with barcode B
In-tissue ligation with barcode B
10m
10m
Follow the steps from 50 to step 57, and only need to replace the barcode A with barcode B..
Image the tissue slide using Keyence microscopy with an 10× objective lens.
Lysis
Lysis
Prepare the reverse crosslinking buffer (for one tissue slide)
5 µL 1M Tris-HCl,pH 8.0
10 µL 10mM EDTA,pH 8.0
10 µL 10% SDS
4 µL 5M NaCl
2 µL 20mg/ml proteinase K
69 µL Nuclese-free water
Attach the PDMS reservoir and 12mm diameter clamp to the tissue.
Add 100 µL reverse crosslinking buffer and cover the hole with Parafilm or tapes.
Incubate the tissue slide at 60 °C for 02:00:00 .
2h
Collect the lysate into a fressh PCR tube and Incubate the tissue slide at 60 °C forOvernight .
Pause point Store lysate at −80 °C.
Purify the lysate with Zymo DNA Clean and concentrator kit in a 5:1 ratio (sample : buffer).
Elute the purified DNA with 100 µL RNase free water.
gDNA and cDNA separation
gDNA and cDNA separation
10m
10m
Prepare buffer:
Wash 40 µL Dynabeads‱ MyOne‱ Streptavidin C1 beads with 800 µL 1XTBW buffer.
Repeate step 68 for 2 times.
Resuspend the beads with 100 µL 2xBW buffer and the purified DNA from step 66.
Agitate the DNA-beads at Room temperature for 01:00:00 .
1h
Use a magnetic rack to separate beads (containing cDNA/ADT) and supernatant (containing gDNA).
RNA/ADT library construction
RNA/ADT library construction
Add 400 µL of 1X TBW buffer to the seperated beads and resuspend.
Separate the beads from the supernatant on the magnetic rack for 1 min and remove the supernatant.
Repeate step 73-74.
Add 400 µL of 1XTris-HCl buffer to the seperated beads and resuspend.
Agitate beads for 00:05:00 at Room temperature .
5m
Separate the beads from the supernatant on the magnetic rack for 1 min and remove the supernatant.
Add 400 µL of RNase free water to the seperated beads.
Prepare TSO solution
22 µL 10mM dNTPs
44 µL 5X Maxima RT buffer
44 µL 20% Ficoll PM-400 solution
5.5 µL 100 micromolar (µM) TSO primer
11 µL Maxima H Minus Reverse Transcriptase
5.5 µL RNase Inhibitor
88 µL RNase free water
Separate the beads from the supernatant on the magnetic rack for 1 min and remove the supernatant.
Add 200 µL TSO solution and gentlely shake the beads at Room temperature for 00:30:00
30m
Gentlely shake the beads at 42 °C for 01:30:00
1h 30m
Separate the beads from the supernatant on the magnetic rack for 1 min and remove the supernatant.
Add 400 µL of 1XTris-HCl buffer to the seperated beads and resuspend.
Agitate beads for 00:05:00 at Room temperature .
5m
Separate the beads from the supernatant on the magnetic rack for 1 min and remove the supernatant.
Add 400 µL of RNase free water to the seperated beads.
Prepare the PCR mixture
110 µL 2X KAPA HIFI HotStart Master Mix
8.8 µL 10 micromolar (µM) Primer 1
8.8 µL 10 micromolar (µM) Primer 2
1 µL 10 micromolar (µM) Primer 3
91.4 µL Nuclease Free water
Add 200 µL PCR mix to the separated beads. Splite the beads into 4 PCR tubes.
PCR thermocyling was performed as following:
Separate the beads from the supernatant on the magnetic rack for 1 min and transfer the supernant to a fresh tube (1.5 ml).
Add 10 µL 20X EvaGreen and splite into 5 qPCR tubes (46 µL for each tube).
Run with a qPCR machine with the following thermocycling conditions:
Once the qPCR signal began to plateau, reactions are stopped.
PCR reactions are purified using a 0.6x ratio of SPRI beads according to the manufacturer's protocol, and the cDNA is eluted with 20 µL Nuclease free water.
Note
Keep the supernatant for ADT library construction.
Confirm the quality and quantity of the cDNA library with the agilent tapestation.
Note
The sample can be stored at -20 °C
RNA library construction
Nextera XT DNA Library Preparation Kit is used for final RNA library construction.
ADT library construction
During SPRI cleanup (Step 95), the supernatant is saved and the short DNA derived from antibody oligos is purified with 2.0× SPRI beads:
- Transfer the supernatant to a new tube and add 1.4X SPRIselect reagent to obtain a final SPRI volume of 2X SPRI.
- purify the SPRI beads according to the manufacturer's protocol, and the beads are eluted with 50 µL Nuclease free water.
- Second time purification with 2.0X SPRI beads and he beads are eluted with 30 µL Nuclease free water.
ADT library amplification:
Prepare 100ul PCR reaction with purified ADT:
- 30 µL purified ADT fraction
- 50 µL 2x KAPA Hifi PCR Master Mix.
- 1.5 µL 10 micromolar (µM) Nextera DNA Index.
- 1.5 µL 10 micromolar (µM) P5 oligo.
- 17 µL Nuclease free water.
PCR program:
95˚C 3 min
95˚C 20 sec |
60˚C 30 sec | ~ 6-10 cycles
72˚C 20 sec |
72˚C 5 min
PCR reactions are purified using a 1.6x ratio of SPRI beads according to the manufacturer's protocol, and the cDNA is eluted with 20 µL Nuclease free water.
Confirm the quality and quantity of the cDNA library with the agilent tapestation.
The sample can be stored at -20 °C
ATAC/nano-CUT&Tag library construction
ATAC/nano-CUT&Tag library construction
During Step 72, the supernatant is saved and the gDNA is purified with Zymo DNA Clean and concentrator kit in a 5:1 ratio (sample : buffer).
The column is eluted with 45 µL Nuclease free water.
ATAC/nano-CUT&Tag library amplification:
Prepare 100ul PCR reaction with purified ADT:
- 45 µL purified ATAC/nano-CUT&Tag fraction
- 50 µL 2X NEB Master Mix.
- 2.5 µL 10 micromolar (µM) Nextera DNA Index.
- 2.5 µL 10 micromolar (µM) P5 oligo.
PCR program:
58˚C 5 min
72˚C 5 min
98˚C 30 sec
98˚C 10 sec |
60˚C 30 sec | ~ 13 cycles
72˚C 5 min
PCR reactions are purified using a 1.2x ratio of SPRI beads according to the manufacturer's protocol, and the final gDNA library is eluted with 20 µL Nuclease free water.
Confirm the quality and quantity of the library with the agilent tapestation.
The sample can be stored at -20 °C