A | B | C | |
material | vendor | catalog number | |
UltraPure™ DEPC-treated Water 1L | ThermoFisher Scientific | 750023 | |
Pierce™ 16% Formaldehyde (w/v), Methanol-free | ThermoFisher Scientific | 28908 | |
PBS - Phosphate-Buffered Saline (10X) pH 7.4, RNase-free | ThermoFisher Scientific | AM9624 | |
Ethyl alcohol, Pure | Sigma-Aldrich | E7023 | |
Triton™ X-100 solution | Sigma-Aldrich | 93443 | |
pepsin | Sigma-Aldrich | 10108057001 | |
SuperScript™ IV Reverse Transcriptase | ThermoFisher Scientific | 18090010 | |
Advantage® UltraPure dNTP Combination Kit | ClonTech | 639132 | |
RNase inhibitor | Enzymatics | Y9240L | |
Aminoallyl dUTP, 4 mM in TE buffer *UltraPure Grade*, Anaspec Inc | Anaspec (VWR) | AS-83203 | |
BS(PEG)9 | ThermoFisher Scientific | 21582 | |
UltraPure™ DNase/RNase-Free Distilled Water | TheroFischer Scientific | 10977023 | |
Ribonuclease; RNAse H; Conc. 5,000 U/mL; 5,000 U; incl. 10X buffer | Fisher Scientific | 50305945 | |
RNase Cocktail™ Enzyme Mix | Invitrogen | AM2288 | |
Ampligase® Enzyme and Buffer | VWR | 76081-598 | |
SSC (20X), RNase-free | ThermoFisher Scientific | AM9763 | |
Formamide (Deionized) | ThermoFisher Scientific | AM9342 | |
BSA, Molecular Biology Grade | NEB | B9000S | |
Phi29 DNA polymerase (10U/uL) | ThermoFisher Scientific | EP0094 | |
Acryloyl-X, SE in DMSO | ThermoFisher Scientific | A20770 | |
Acrylamide/Bis-acrylamide, BioReagent, for molecular biology, 37:1 (ratio) | Sigma-Aldrich | A6050-100ML | |
Ammonium persulfate | Sigma-Aldrich | A3678 | |
TEMED | Fisher Scientific | 17919 | |
gel slick solution | Lonza | 50640 | |
TrueBlack lipofuscin autofluorescence quencher | Biotium | 23007 | |
Silicone Isolators JTR20R-2.5 20mm DIA x 2.4 mm Depth 25 x 25mm OD No PSA | Grace Bio-Labs | 664304 | |
Coverslips, Glass, 18mm dia. | Ted Pella | 260369 | |
Azer Scientific cover glass, No. 1.5, 24 x 60 mm | Neta Scientific | Azer-1152460 |
A | B | C | |
oligo name | vendor | sequence | |
5N_dc10-Cy5_N9 | IDT | /5AmMC12/CCGATAGTCACGATCTGTGGNNNNNNNN*N | |
dT20_dc7-488 | IDT | CATGGATTCGCGGAGGATCATTTTTTTTTTTTTTTTTTT*T | |
FISSEQ_ppRCA primer | IDT | GATATCGGGAAGCTGA*A*G | |
DARTFISH_anchor_Cy3 | IDT | /5Cy3/CTTCAGCTTCCCGATATCCG | |
dcProbe7-AF488 | IDT | /5Alex488N/TGATCCTCCGCGAATCCATG | |
dcProbe10-ATTO647N | IDT | /5ATTO647NN/CCACAGATCGTGACTATCGG | |
dcProbe0-AF488 | IDT | /5Alex488N/TGTATCGCGCTCGATTGGCA | |
dcProbe0-Cy3 | IDT | /5Cy3/CGTATCGGTAGTCGCAACGC | |
dcProbe0-ATTO647N | IDT | /5ATTO647NN/ACGCTACGGAGTACGCCACT | |
dcProbe1-AF488 | IDT | /5Alex488N/TCTTGCGTGCGATACGGAGT | |
dcProbe1-Cy3 | IDT | /5Cy3/AACGGTATTCGGTCGTCATC | |
dcProbe1-ATTO647N | IDT | /5ATTO647NN/CTGGTTCGGGCGTACCTAAC | |
dcProbe2-AF488 | IDT | /5Alex488N/AGAACTTGCGCGGATACACG | |
dcProbe2-Cy3 | IDT | /5Cy3/CTACTTCGTCGCGTCAGACC | |
dcProbe2-ATTO647N | IDT | GACGAACGGTCGAGATTTAC/3ATTO647NN/ | |
dcProbe3-AF488 | IDT | /5Alex488N/GAATTGTCCGCGCTCTACGA | |
dcProbe3-Cy3_2 | IDT | /5Cy3/TCGTACTTCGACGGCACTCA | |
dcProbe3-ATTO647N | IDT | /5ATTO647NN/AACTGCGACCGTCGGCTTAC | |
dcProbe4-AF488 | IDT | /5Alex488N/CGGAATACGTCGTTGACTGC | |
dcProbe4-Cy3 | IDT | /5Cy3/TACCATTCGCGTGCGATTCC | |
dcProbe4-ATTO647N_2 | IDT | /5ATTO647NN/ACTCTACCGGCAATCGCGTC | |
dcProbe5-AF488 | IDT | /5Alex488N/GAGTGTCGCGCAACTTAGCG | |
dcProbe5-Cy3 | IDT | /5Cy3/ACGTCTGCGTACCGGCTTAG | |
dcProbe5-ATTO647N | IDT | /5ATTO647NN/CATGCGATTAACCGCGACTG | |
dcProbe6-AF488_2 | IDT | /5Alex488N/CTTGCGGCGACAGTCGAACA | |
dcProbe6-Cy3 | IDT | /5Cy3/TCGTAACCCGTGCGAAGTGC | |
dcProbe6-ATTO647N | IDT | /5ATTO647NN/CTCTCGTAGCGTGCGATGAG | |
dcProbe7-AF488_2 | IDT | /5Alex488N/TTAGGTCGCCTACCGACTGC | |
dcProbe7-Cy3 | IDT | /5Cy3/GCCACATCGACTCGGTCTAT | |
dcProbe7-ATTO647N | IDT | GCTCAGCCGGACGAGTAGAT/3ATTO647NN/ |
A | B | |
component | volume (mL) | |
DEPC-water | 52 | |
16% PFA | 20 | |
10X PBS | 8 |
A | B | |
component | x1 volume (uL) | |
DEPC-water | 875 | |
10X PBS | 100 | |
10% Triton X-100 | 25 |
A | B | |
component | x1 volume (uL) | |
1% pepsin | 1 | |
0.1N HCl | 99 |
A | B | |
component | x1 volume (uL) | |
DEPC-water | 90.75 | |
5X SSIV buffer | 30 | |
10mM dNTP | 3.75 | |
100uM N5_dc10-Cy5_N9 | 3.75 | |
100uM dT20_dc7-488 | 3.75 | |
0.1M DTT | 7.5 | |
4mM aminoallyl-dUTP | 1.5 | |
RNase inhibitor (40U/uL) | 1.5 | |
SuperScript IV Reverse Transcriptase | 7.5 | |
total | 150 |
A | B | |
component | x1 volume (uL) | |
250mM BS(PEG)9 | 10 | |
10X PBS | 50 | |
ultrapure water | 440 |
A | B | |
component | x1 volume (uL) | |
ultrapure water | 168 | |
10X RNase H buffer | 20 | |
RNase H (5U/uL) | 10 | |
RNase Cocktail | 2 |
A | B | |
component | x1 volume (uL) | |
ultrapure water | 98.2 | |
10X Ampligase buffer | 15 | |
HB_Feb2022 probes ( 27.2 ng/uL) | 25.3 | |
100nM PLP1 oligos | 1.5 | |
Ampligase (5U/uL) | 10 |
A | B | |
component | x1 volume (uL) | |
ultrapure water | 119 | |
20X SSC | 20 | |
formamide | 60 | |
100 uM FISSEQ_ppRCA primer | 1 |
A | B | |
component | x1 volume (uL) | |
ultrapure water | 119.25 | |
10X Phi29 polymerase buffer | 15 | |
10mM dNTP | 3.75 | |
4mM aminoallyl-dUTP | 1.5 | |
NEB BSA (20mg/mL) | 7.5 | |
ThermoFisher Phi29 polymerase | 3 | |
total | 150 |
A | B | |
Component | x1 volume (uL) | |
10X PBS | 50 | |
10mg/mL Acryloyl-X, SE in DMSO | 10 | |
ultrapure water | 440 |
A | B | |
Component | x1 volume (uL) | |
10X PBS | 50 | |
40% Acrylamide/Bis (37:1) | 50 | |
ultrapure water | 400 |
A | B | |
component | x1 volume (uL) | |
Acrylamide solution | 138 | |
4% APS | 6 | |
5% TEMED | 6 |
A | B | |
component | x1 volume (uL) | |
100 uM dcProbe0_AF488 | 1 | |
100 uM dcProbe0_Cy3 | 1 | |
100 uM dcProbe0_ATTO647 | 1 | |
100% formamide | 60 | |
20X SSC | 20 | |
ultrapure water | 117 |