Preparation of Fresh Frozen Sections
1 Crush dry ice into pieces of 2-3 cm in size
2 Put the crushed dry ice lumps into a 100 ml beaker, then fill it with iso-pentane
3 Put the dissected tissues into an embedding container filled with O.C.T compound
4 Flash-freeze the tissues by submerging into the iso-pentane cooled with dry ice.
5 Make the sections at a thickness of 10 μm with a cryostat and mount them on MAS-coated glass slides (Matsunami).
Note: Subsequent processing can be facilitated by creating the hydrophobic barriers around the sections with a pap pen facilitates
Fixation and permeabilization
6 Wash the tissue sections twice with PBS to remove the O.C.T compound
7 Fix the sections with 4% PFA in PBS for 10 min at room temperature
8 Wash the sections twice with PBS
9 Incubate the sections for 3 min with PBS containing 3% Triton X-100 at room temperature
10 Wash the sections twice with PBS
11 Incubate the sections with 0.1N HCl in nuclease-free water for 5 min at room temperature
12 Remove the supernatant
13 Incubate the sections with 1M Tris-HCl (pH8.0) for 5 min at room temperature
14 Wash the sections twice with PBS
15 Immerse the sections in PBS preheated to 65°C in a staining jar and incubate in a hybridization oven for 5min
16 Immerse the sections in a staining jar containing chilled PBS
In situ RT
17 Mix 0.5 μl of 500 ng/μl caged Oligo DNA, 0.5 μl of 10 mM dNTPs, and 5 μl of nuclease-free water in a 0.2 ml tube, and heat to 65°C and then quickly cool to 4°C using Thermal cycler
18 Add 2 μl of 5× First Strand Buffer, 1 μl of 0.1 M DTT, 0.5 μl of 40 U/μl RNase Out, and 0.5 μl of 200 U/μl Superscript II Reverse Transcriptase to the primer mix prepared above (17)
19 Drop 10 μl of the RT mix prepared above (18) onto the section and seal with a coverslip
20 Incubate the sections at 42 °C for 1 hr in a PBS-humidified chamber
21 Immerse the sections in PBS preheated to 70 °C in a staining jar and keep at 70 °C in a hybridization oven for 10min
22 Immerse the sections in a staining jar cold containing chilled PBS
23 Wash the sections twice with PBS
Option : Immunostaing
24 Incubate the sections for 15 min with blocking solution at room temperature
25 Incubate the sections with primary antibodies diluted in blocking solution for 2 hr at room temperature or overnight at 4 °C in a hybridization oven
26 Wash the sections three times with TBST
27 Incubate the sections with 2nd antibodies diluted in blocking solution for 1 hr at room temperature or overnight at 4 °C in a hybridization oven
28 Wash the sections three times with TBST
29 Incubate the sections with Nuclear Violet LCS (1:1000) with blocking solution for 15 min at room temperature in a hybridization oven
30 Mount the sections with SlowFade Diamond before coverslipping
UV irradiation
31 Irradiate 365 nm-centered UV light to the ROIs for 15 min
Note: Since the caged compound (NPOM) used in PIC is cleaved most efficiently at 365 nm, the irradiation can be performed with the fluorescence filter cube that are conventionally used to excite nuclear staining dyes with blue fluorescence such as DAPI and Hoechst. We used Leica A filter cube (#11513873; 340–380 nm excitation) for uncaging under a Leica DM5000 B fluorescence microscope illuminated with an EL6000 100 W Hg lamp.
Cell lysis
32 Strip the cover slip, and wash the sections three times with PBS
33 Drop 40 μ l of proteinase K lysis solution to the sections
34 Incubate the sections in a hybridization oven at 42 °C for 30 min
35 Scrape the sections and collect the cell lysates and debris in a 1.5 ml tube
36 Drop 40 μl of proteinase K lysis solution to the section, again
37 Incubate the sections in a hybridization oven at 42 °C for 30 min, again
38 Collect the cell lysates and debris in the same tube as (36) (total volume ≃ 80 μl)
39 Incubate for 30 min at 55 °C
40 Purify cDNA:mRNA hybrids with Qiagen MinElute PCR Purification kit
41 Elute with 15 μl of nuclease-free water
Second-strand DNA synthesis
42 Mix the eluted 15 μl of cDNA:mRNA hybrids with 2 μl of 5xFirst Strand Buffer, 2.31 μl of 5x Second Strand Buffer, 0.23 μl of 10 mM dNTPs, 0.08 μl of E.coli DNA ligase (10 U/μl), 0.3 μl of E.coli DNA polumerase (10 U/μl) and 0.08 μl of E.coli RNaseH (2 U/μl)
43 Incubate for 2 hr at 16 °C
44 Mix 20 μl of second-strand DNA sample and 24 μl of Ampure binding buffer mix (Ampure XP beads : Beads binding buffer = 1 : 5) and vortex thoroughly
45 Incubate for 15 min at room temperature
46 Place the sample tube on the magnetic stand and incubate for 5 min at room temperature
47 Remove the supernatant
48 Add 200 μl of 80% Ethanol and incubate for 30 sec at room temperature
49 Remove the supernatant
50 Add 200 μl of 80% Ethanol and incubate for 30 sec at room temperature
51 Remove the supernatant
52 Spin down the sample tube
53 Place the sample tube on the magnetic stand and incubate for 2 min at room temperature
54 Remove the supernatant and air dry
55 Suspend the beads with 6.4 μl of nuclease-free water by pipetting
in virto transcription
56 Mix the 6.4 μl of beads suspension containing double-strand cDNAs with 1.6 μl of ATP, 1.6 μl of GTP, 1.6 μl of CTP, 1.6 μl of UTP, 1.6 μl of 10xT7 reaction buffer, and 1.6 μl of T7 Enzyme from the MEGAscript T7 Transcription Kit
57 Incubate for 17 hr at 37 °C
58 Add 1 μl of TURBO DNase from the MEGAscript T7 Transcription Kit and incubate for 15 min at 37 °C
59 Add 3 μl of ExoSAP-IT Express PCR Product Cleanup and incubate for 5 min at 37 °C
60 Add 5.5 μl of fragmentation Buffer and incubate for 3 min at 94 °C and quickly cool to 4°C using Thermal cycler
61 Place the sample tube on ice and add 2.75 μl of 0.5 M EDTA pH8.0
62 Place the sample tube on the magnetic stand and incubate for 5 min at room temperature
63 Collect 28 μl of supernatant in a new 0.2 ml tube
aRNA Cleanup
64 Mix 28 μl of aRNA samples with 50.4 μl of RNAClean XP beads and vortex thoroughly
65 Incubate for 10 min at room temperature
66 Place the sample tube on the magnetic stand and incubate for 5 min at room temperature
67 Remove the supernatant
68 Add 200 μl of 70% Ethanol and incubate for 30 sec at room temperature
69 Remove the supernatant
70 Add 200 μl of 70% Ethanol and incubate for 30 sec at room temperature
71 Remove the supernatant
72 Spin down the sample tube
73 Place the sample tube on the magnetic stand and incubate for 2 min at room temperature
74 Remove the supernatant and air dry
75 Suspend the beads with 7 μl of nuclease-free water by pipetting
76 Place the sample tube on the magnetic stand and incubate for 5 min at room temperature
77 Collect 5 μl of supernatant in a new 0.2 ml tube
Library preparation
78 Mix eluted 5 μl of aRNAs with 1 μl of RandomhexRT (250 ng) and 0.5 μl of 10 mM dNTPs
79 Heat the tubes at 65°C and then quickly cool to 4°C using Thermal cycler
80 Add 2 μl of 5× First Strand Buffer, 1 μl of 0.1 M DTT, 0.5 μl of 40 U/μl RNase Out, and 0.5 μl of 200 U/μl Superscript II Reverse Transcriptase
81 Incubate for 10 min at 25°C, then for 1 hr at 42°C
82 Add 8.4 μl of nuclease-free water, 1.8 μl of RNA PCR primer 1 (10 μM), 1.8 μl of RNA PCR primer 2 (10 μM) and 22.5 μl of Pusion High-Fidelity PCR Master Mix
83 Amplify by PCR (98°C for 30 s, followed by 11 cycles of 98°C for 10 s, 60°C for 30 s and 72°C for 30 s, with final extension at 72°C for 10 min)
84 Add 1 μl of RNaseA (10 mg/ml) and incubate for 30 min at 37 °C
85 Add 6 μl of nuclease-free water and 32.5 μl Ampure XP beads, and vortex thoroughly
86 Incubate for 15 min at room temperature
87 Place the sample tube on the magnetic stand and incubate for 5 min at room temperature
88 Collect the supernatant in a new 0.2 ml tube
89 Add 12.5 μl Ampure XP beads, and vortex thoroughly
90 Incubate for 10 min at room temperature
91 Place the sample tube on the magnetic stand and incubate for 5 min at room temperature
92 Remove the supernatant
93 Add 200 μl of 80% Ethanol and incubate for 30 sec at room temperature
94 Remove the supernatant
95 Add 200 μl of 80% Ethanol and incubate for 30 sec at room temperature
96 Remove the supernatant
97 Spin down the sample tube
98 Place the sample tube on the magnetic stand and incubate for 2 min at room temperature
99 Remove the supernatant and air dry
100 Suspend the beads with 32 μl of nuclease-free water by pipetting
101 Incubate for 2 min at room temperature
102 Place the sample tube on the magnetic stand and incubate for 5 min at room temperature
103 Collect 30 μl of supernatant in a new 0.2 ml tube
104 Add 19.5 μl Ampure XP beads, and vortex thoroughly
105 Incubate for 15 min at room temperature
106 Place the sample tube on the magnetic stand and incubate for 5 min at room temperature
107 Collect the supernatant in a new 0.2 ml tube
108 Add 7.5 μl Ampure XP beads, and vortex thoroughly
109 Incubate for 10 min at room temperature
110 Place the sample tube on the magnetic stand and incubate for 5 min at room temperature
111 Remove the supernatant
112 Add 200 μl of 80% Ethanol and incubate for 30 sec at room temperature
113 Remove the supernatant
114 Add 200 μl of 80% Ethanol and incubate for 30 sec at room temperature
115 Remove the supernatant
116 Spin down the sample tube
117 Place the sample tube on the magnetic stand and incubate for 2 min at room temperature
118 Remove the supernatant and air dry
119 Suspend the beads with 17.5 μl of nuclease-free water by pipetting
120 Place the sample tube on the magnetic stand and incubate for 5 min at room temperature
121 Collect 15 μl of supernatant in a new 0.2 ml tube
122 Quantify cDNA library using high-sensitivity DNA chips on a Bioanalyzer 2100 and Library Quantification Kit
123 Sequence with Illumina sequencer (1–3 million reads per sample)
124 Analyze the sequence data according to CEL-seq2 protocol (Hashimshony, T. et al. Genome Biol 2016)