CLARITY Protocol

100 um

DAY1 晚上

RNA anchoring（一个晚上）

  1. PBS溶液洗2×10 min，1× MOPS（50 mM）清洗20 min。

  2. mephalen X（2mg/mL）用1× MOPS（50 mM）稀释到工作mephalen X（1mg/mL，指Label IT浓度）。

  1. 将slice与工作mephalen X溶液在37℃下，反应overnight

  2. 用PBS溶液清洗2×10 min

DAY2上午

  1. then incubated with monomer buffer (4% acrylamide, 0.2% bis-acrylamide, 2X SSC) for 30min at r.t.（1h at 4℃）

10 ml 2X SSC (10g) + Acrylamide (400 mg) + bis-acrylamide (20 mg)

  1. The buffer was aspirated and 500 µL of polymerization mixture (0.2% ammonium persulfate, 0.2%tetramethylethylenediamine dissolved in monomer buffer) was added to the center of the sample, which were immediately covered by Gel Slick coated coverslip and incubated for 1 hour at r.t.,
  1. The tissue-gel hybrids were digested with Proteinase K (0.2 mg/ml) at 37℃ for overnight

DAY2 晚上

  1. 24孔板 PBST 20min*3

  2. 2 ml tube

Block (25℃，反应30 min)

Reagent Stock Final 100 400
Ddw 57.5 230
Ampligase 10× 1× 10 40
KCl 1M 0.05M 5 20
Formamide 100% 20% 20 80
oligo-dT 10 uM 0.1 uM 1 4
BSA 10 ug/uL 0.2 ug/uL 2 8
t-RNA 10 ug/uL 0.2 ug/uL 2 8
Ribolock（酶） 40 U/uL 1 U/uL 2.5 10
  1. Hybridization master mix的配置
Reagent Stock Final 100 ul 400
Ddw 0 0
Ampligase buffer 10× 1× 10 40
KCl 1M 0.05M 5 20
Formamide 100% 20% 20 80
Probe 10 uM 0.2 uM 58.5 234
BSA 10 ug/uL 0.2 ug/uL 2 8
t-RNA 10 ug/uL 0.2 ug/uL 2 8
Ribolock（酶） 40 U/uL 1 U/uL 2.5 10

烘箱55℃ 15 min，45℃ 1 day

  1. Wash buffer. 3*20 min

0.05 % PBST 3*5 min

DAY3 晚上

  1. Ligation solution
Reagent Stock Final 100 400
RNase-free water 75.5 302
SplintR buffer 10× 1× 10 40
BSA 10 ug/uL 0.2 ug/uL 2 8
SplintR（酶） 10 ug/uL 0.2 ug/uL 10 40
Ribolock（酶） 40 U/uL 1 U/uL 2.5 10

盖上PDMS小盖子，37 ℃,overnight

PBST 2×10 min

DAY4 上午

  1. RCA
Reagent Stock Final 100 ul 400
Ddw 57 228
NH2-dUTP 2 mM 50 uM 2.5 10
φ29 buffer 10× 1× 10 40
dNTP 10 mM 250 uM 2.5 10
Glycerol 50% 5% 10 40
BSA 10 ug/uL 0.2 ug/uL 2 8
RCA primer 10 uM 0.6 uM 6 24
φ29（thermo） 10 U/uL 0.5 U/uL 2 8

RCA一天

DAY5

  1. 固定（2.5h）

PBST 0.05% 2×5 min

2% BS(PEG9) in PBST 1 h R.T.

PBST 0.05% 3×5 min

65%甲酰胺 3×10 min

PBST 0.05% 2×5 min

  1. 染色 （3.5h）

probe的混合液 2h

WB 3*10 min

PBST 3×5 min

DAPI 20 min

PBST 2*5 min