The libraries are made after the 10x Protocols (RNA v3.1, ATAC v1.1, Multiome, HT)
After they finished they are measured with Qubit for quantity check and Fragment Analyzer (FA) for quality check.
You find the libraries in the library-boxes in the -20°C Freezer (Every protocol has their separate drawer which contains the reagents and also Boxes for libraries, dilutions and pools.)
Before you start picking the libraries check first if there is already a dilution and if it is still enough for you pool. (around 10µl should be ok)
Put all the libraries/dilutions you need on your rack and bring the Qubit stuff to your bench. Qubit contains: Standard 1 and 2 (fridge in room 147), Working solution kit and Qubit itself (Qubit-drawer in room 149)
For every Pool we have a pooling sheet which is divided in diluting library, measurement of the dilution, pooling the dilutions and the measurement of the pool.
After you made sure which samples had to dilute, you put them in the 1. Table. The grey parts had to be filled and the blue ones are calculated by filling the grey fields.
Make sure you never dilute less then 20µl (you will need this much to measure the dilution and spike, and pool them)
After you mixed the calculated EB-Buffer and the library you vortex it.
Use the Qubit now to check if the concentration is close 10 nM.
For this you need to measure the dilutions with the qubit and fill in the results in the table which calculate the conc.
You will get a feeling which conc. is still ok to continue with but for a first guideline something between 8 and 12nM is still useable.
Now copy the nM concentrations to the empty DNA conc. slot in the Pool-table and mix the pool like it is shown in the blue part of the table.
Make sure that you are doing the sub-pool in a separate tube and add only the amount of the sub pool that is shown on the left side of this blue table part.
Also make sure not forget to enter the EB-Buffer. It is in the end of this table and easy to oversee.
After you mixed and vortex the pool you should measure it again with the qubit.
It is the same procedure with the dilutions. Note the result and type it into the table. It should be around 4nM now.
If it is around 3,5 or 4,5 nM it is still fine. You will get a feeling for this, too.
The finished pool will be stored in the Pool boxes (mostly red; -20°C) in the same freezer you’ve got the libraries and the dilutions from. Every protocol has also their own Pool box to not be confused.
Pools Name:
name the Protocol: SN → RNA; SA → ATAC; MSN → Multiome RNA; MSA → Multiome ATAC
count the pools of the Day: Pool1, Pool2, Pool3….
put the date: ddmmyy → 050822