TITLE: MAS-ISO-seq - from 10x Single Cell Gene Expression Libraries
AUTHORS: Aziz Al'Khafaji

[DESCRIPTION]
Method for MAS-ISO-seq from 10x Single Cell Gene Expression Libraries.

[STEPS]
2. WTA amplification:

Set up the following reactions on ice

For 5' libraries:
 

For 3' libraries:

 


 

3. Reaction cleanup and quantification

0.8x SPRIselect cleanup - 80 µL beads in 100 µL PCR reaction from step 2.
Elute in 46 µL low-TE
Qubit quantification

4. TSO artifact removal


Transfer 10 μL (100 μg) resuspended Dynabeads™ kilobaseBINDER™ streptavidin beads to a PCR tube. 
Place the tube on the magnet for 2 min.
Carefully remove and discard the supernatant while the tube remains on the magnet. Avoid touching the bead pellet with the pipette tip.
Remove the tube from the magnet. Add 40 μL Binding Solution along the inside wall of the tube where the beads are collected and gently resuspend by pipetting. Note: the solution may be viscous. Avoid foaming.
Place the tube on the magnet for 2 min and remove the supernatant.
Resuspend the beads in 40 μL Binding Solution.
Add 40 μL of a solution containing the biotinylated DNA-fragments to the resuspended beads. Mix carefully to avoid foaming of the solution.
Incubate the tube at room temperature for 3 hours on a roller to keep the beads in suspension.
Place the tube on the magnet and remove the supernatant as in step 3, above.
Wash the Dynabeads®/DNA-complex 2x in 80 μL Washing Solution and once in distilled water.
Resuspend the Dynabeads®/DNA-complex in 40ul Low-TE.
Add 2ul USER and incubate in a rotator at 37C for 2 hours.
Place the tube on the magnet and move the supernatant containing the library to a fresh tube.
Cleanup - 0.8x SPRI (32 µL beads in 40 µL library)
Elute in 46 µL low-TE.
Qubit quantification

5. MAS adapter PCR

Set up all reactions on ice

Create the following master mix:
 

2. Distribute 90 µL of Master Mix into each of 15 PCR tubes
3. Distribute 10 µL 5 µM MAS-seq primer pair mix into each of 15 PCR tubes


Cycling conditions: 
 
Note  - optimal cycle number is a function of amount of input material after TSO artifact removal. See table below for cycle determination

 


6. Reaction cleanup

Pool 15 x 95 µL reactions in 5ml tube. (Note* attaining equimolar amounts of each adapter PCR is key to efficient array assembly - be mindful to add the same amount of each adapter PCR to the pool. To account for handling, 95/100 µL of the PCR is advised to be pooled from each reaction.)
SPRIselect cleanup - 0.7x (997.5 µL beads in 1425 µL pooled MAS adapter PCR)
Elute in 450 µL low TE buffer.
Qubit quantify
Move 435 µL of the library to a 1.5 mL microtube tube and added 15 µL of USER enzyme. (Note* 10-15 µg is advised going into this step. Dilute going into this step if sample is too concentrated.)
Incubate reaction at 37 for 2.5 hours
Add 51 µL Hifi Ligase buffer and 15ul Hifi ligase to the USER reaction.
Distribute to 5x PCR tubes and set in thermocycler at 42C for 2 hours.
Pool reaction into a 1.5 mL microtube using a wide bore tip
SPRIselect cleanup - 0.7x ( 361.2 µL beads in 516 µL reaction), mix gently with wide bore tip. Set on rotator for 5 min. 
Incubate at 37C for 10min - elute in 180 µL low TE. 
Qubit quantify 
7. MAS library quantification

Quantify MAS arrays with Genomic DNA ScreenTape or Femto Pulse.

See example below:
 
Optional Size Selection

SMRTbell Express Template Prep: Use this as input material for PacBio SMRTbell Express Template Prep Kit 2.0, starting at the " Remove ssDNA Overhangs" step (page 8: https://www.pacb.com/wp-content/uploads/Procedure-Checklist-Preparing-HiFi-SMRTbell-Libraries-using-SMRTbell-Express-Template-Prep-Kit-2.0.pdf)

Minimum starting material going into the PacBio SMRTbell Express Template Prep Kit 2.0 : 5µg
1. Mix MAS primer pairs:

Order primers reconstituted as 100µM

Mix equal volumes of primer pairs according to the table below to create a 50µM MAS primer mix plate. 

Create a 5µM working MAS-primer plate by diluting 10-fold in low-TE. 

 
   MAS-seq primer mixing strategy  wellsadapter pairs1A1 & C1*A|B2A2 & C2B|C3A3 & C3C|D4A4 & C4D|E5A5 & C5E|F6A6 & C6F|G7A7 & C7G|H8A8 & C8H|I9A9 & C9I|J10A10 & C10J|K11A11 & C11K|L12A12 & C12L|M13B1 & D1M|N14B2 & D2N|O15B3 & D3O|P*