BARseq - high-throughput cell typing with in situ sequencing
Xiaoyin Chen, Anthony M. Zador, Mararue
Abstract
This protocol describes the application of BARseq as a standalone in situ sequencing method to achieve multiplexed interrogation of endogenous genes. In this variation, BARseq is similar to in situ sequencing (ISS), but uses Illuimna SBS for sequencing readout.
Steps
Library preparation
Tissues with barcoded neurons should be cryo-sectioned to 20 μm and mounted on slides. Slides can be stored at -80 °C for up to a month.
DAY 1
Take slide(s) out of -80 °C and immerse immediately in 4% paraformaldehyde in 1x PBS (2 slides per 50mL falcon tube, back-to-back)
Incubate for 1 hour at room temperature on slow shaker
Wash the slides by immersing in 1x PBS (2 slides per 50ml falcon tube, back to back)
Wipe excess PBS off the surface of the chamber, then stick on the Hybriwell-FL chambers. Note that the ports on the chamber should be placed as far away from the tissue slices as possible.
Wash twice in PBST (1x PBS + 0.5% Tween-20)
Wash in 70% Ethanol for 5 mins
Wash in 85% Ethanol for 5 mins
Wash in 100% Ethanol for 5 mins
Replace with new 100% Ethanol, drop extra 100% Ethanol on top of slides and cover with ParaFilm to avoid evaporation. Incubate for at least 1.5 hrs at 4 °C (up to 3 hours)
Wash in PBST for 4-6 times, until all bubbles are cleared in the chamber and PBST flows into and out of the chamber smoothly.
Make reverse transription mix: 50 μM N20 primer (XC2757), 20 U/μL RevertAid H Minus M-MuLV reverse transcriptase, 500 μM dNTP, 0.2 μg/μL BSA, 1 U/μL RiboLock RNase Inhibitor, 1x RevertAid RT buffer.
Incubate in reverse transcription mix overnight at 37 °C. Create a humidity chamber to avoid the slides drying out using kim-wipes and DI water.
DAY 2:
Wash with PBST once
Incubate in a mixture of 1μL BS(PEG)9 per 4 μL PBST (e.g. 200ul BS(PEG)9 and 800ul PBST) for one hour at room temperature
Wash with 1M Tris pH 8.0, then incubate in new 1M Tris pH 8.0 for 30 mins
Wash twice in PBST
Make ligation mix: 1x Ampligase buffer, 100 nM padlock probe each, 0.5 U/μL Ampligase, 0.4 U/μL RNase H, 1 U/μL RiboLock RNase Inhibitor, 50 mM KCl (extra of those already provided by the ampligase buffer), 20% formamide.
Incubate in ligation mix for at least 30 mins at 37 °C (can go longer but not shorter), then at least 45 mins at 45 °C (can go longer but not shorter).
Wash twice in PBST
Make RCA mix: 1 U/μL phi29 DNA polymerase, 1x phi29 polymerase buffer, 0.25 mM dNTP, 0.2 μg/μL BSA, 5% glycerol (extra of those from the enzymes), 125 μM aminoallyl dUTP
Incubate in RCA mix overnight at room temperature
DAY 3:
Wash with PBST once
Incubate in a mixture of 1μL BS(PEG)9 per 4 μL PBST (e.g. 200ul BS(PEG)9 and 800ul PBST) for one hour at room temperature
Wash with 1M Tris pH 8.0, then incubate in new 1M Tris pH 8.0 for 30 mins
Wash twice in PBST
Sequencing
Hybridization of Gene sequencing primer:
Wash with FISH wash (2x SSC with 10% formamide)
Hybridize sequencing primer (YS220) with a primer concentration of 1 μM in FISH wash for 10 mins at room temperature
Wash with FISH wash three times, 2 mins each
Wash with PBST twice
Sequence first cycle:
Do the following incubations. Unless noted with incubation temperature, each step is performed at room temperature. For steps without incubation time, treat these as quick washes. large flat metal blocks can be used to place sample slides to quickly cycle through high and low temperatures. This version uses MiSeq Nano v2 kit:
Incorporation Buffer 60 °C 3 mins x1
2% PBST x1
Idoacetamide blocker: For 9.3mg vial dilute pellet in between 2.5-3.5mL 2% PBST. Make fresh tube daily and store out of light.
Idoacetamide blocker 60 °C 3 mins x1
2% PBST x1
Incorporation Buffer x2
IRM 60 °C 3 mins x2
2% PBST x1
2% PBST 60 °C 3 mins x4
Replace 2% PBST with USM and Image if slides are dirty, clean with 70% Ethanol before adding USM
Sequence subsequent cycles: Do the following incubations. Unless noted with incubation temperature, each step is performed at room temperature. For steps without incubation time, treat these as quick washes. large flat metal blocks can be used to place sample slides to quickly cycle through high and low temperatures.
Incorporation buffer x2
2% PBST 60 °C 3 mins x4
Replace 2% PBST with USM and image if slides are dirty, clean with 70% Ethanol before adding USM
CRM 60 °C 3 mins x2
Incorporation buffer x1 - Wipe ports after adding the incorporation buffer, to ensure that no CRM is left on the slide's surface
2% PBST x1
Idoacetamide blocker: For 9.3mg vial dilute pellet in between 2.5-3.5mL 2% PBST. Make fresh tube daily and store out of light.
Idoacetamide blocker 60 °C 3 mins x1
2% PBST x1
Incorporation buffer x2
IRM 60 °C 3 mins x2
2% PBST x1
Hybridization cycle
Hybridize probes:
Make strip buffer: 60% formamide 2xSSC 0.01% Tween20
Strip buffer 60 ˚C 5 mins x3
Cool down quickly on metal plates between washes, place on metal plates in 60 ˚C oven to heat up quickly.
FISH wash (2x SSC with 10% formamide) 1x
Hybridize probes (YS221, XC2758, XC2759, XC2760) with a primer concentration of 1 μM in FISH wash at 60 ˚C for 2 minutes, then for 10 mins at room temperature. Rotate plates in holder to ensure they cool down slowly.
FISH wash x1
0.002 mg/ML DAPI in 2% PBST, room temperature for 5 mins
Replace PBST with USM and image if slides are dirty, clean with 70% Ethanol before adding USM
