Before starting

Documentation

Sequencing Guidelines

Normalize and Pool

1. If not already completed, quantify each of your libraries on Qubit. For most libraries, using the HS dsDNA Qubit assay with 2uL of input will yield a reading. Record the concentration in ng/uL for each library.
2. If not already completed, run your libraries on Tapestation with the HSD1000 assay. Remember to allow Tapestation reagents to sit at room temperature for at least 30 minutes before use. Save your Tapestation results by going to File -> Create Report -> Save as pdf. This file can then be emailed or uploaded to Asana. For the base pair length, we usually use the value of the peak identified by the Tapestation analysis software. This value is shown both on the tracing itself and in the Peak Table for each sample.
3. Download our 10X nM Conversion Calculator here. Enter the concentration (from Qubit) and the base pair length (from Tapestation) in the appropriate cells and it will give you the nM concentration for each library. You also need the Target Cell Recovery values. Normalize and pool all your libraries to 4, 2, 1, or 0.5 nM in a LoBind microcentrifuge tube. If you need to dilute your libraries, we recommend using at least 2uL to minimize pipetting errors. The example sheet of the calculator provides further detail.
4. 10x recommends a minimum of 20,000 read pairs/cell for the RNA expression libraries. The ADT libraries need 5,000 reads/cell, unless doing a 100 or greater ADT panel, which needs 10,000 reads/cell. The HTO libraries need 500 reads/cell. These can all be sequenced on the same run on the NextSeq or the same lane on other sequencers. BioLegend recommends sequencing the RNA expression library at 90% or the run/lane and ADT at 5-10%.
5. Quantify your pool on Qubit and enter into the calculator sheet to check that your pool is close to the nM concentration you normalized to.

Setup Run in Basespace

1. Sign into Basespace, then go to the Prep tab, Biological Samples, and select Import Samples on the upper right. Use Illumina’s Sample Import Template, but only create a single sample for your pool. The SampleID and Name can be the same, but make sure they are unique for each sample. Species can be left blank. Upload the completed .csv to import your samples.
2. Continue to Prep Libraries. This library prep requires a custom setting that gives the samples a single dummy index. On our Basespace account, select the library prep kit “10X Test” from the list. The your project name as the Plate ID. For each sample, check the box next to it on the left, then drag the sample name to the appropriate index well.
3. Proceed to Pool Libraries. Select all your samples on the left, then drag and drop in the pool on the right. Name the pool your project name.
4. Continue to Plan Run. Select NextSeq and name your run your project name. Select Single Read. See table below for cycle selection based on 150 and 75 cycle sequencing kits. If you have run data from samples run on a 150 cycle kit, follow the instructions here to determine if you can use a 75 cycle kit.

1. Select override indexing and enter a single index 1 that is 8 cycles long.
2. Press Sequence to complete planning the run. The run will now be available for selection on the sequencer.

Loading the Sequencer

• These run settings do not generate automatically demultiplexed FASTQ files. 10X recommends downloading the .bcl files and using Cell Ranger to demultiplex. More information can be found here and here.