Table 4 |.

Troubleshooting table

Step	Problem	Possible reason	Solution
1Ai	>25% of cells have no productive clonotype	Misalignment of the sorter so that cells are not being sorted to the center of the wells	Ensure the sorter is properly aligned and that the plate is put squarely in place. Alignment can be checked using fluorescent beads or by placing parafilm across a plate and seeing that sorted droplets are in the center of each well. Rodrigues and Monard49 describe a method using microspheres that can be used to validate sorter alignment.
1Ai	>25% of cells have no productive clonotype plus >25% have alpha only or beta only	Dormant or dying cells	If thawing frozen cells, let cells recover longer before sorting. Be sure to include a viability marker in the sorting.
1A(vii-xxxiv)	>10% of cells have multiple clonotypes	Cross-well contamination prior to pooling	Scrupulous attention to detail during pipetting. A particularly vulnerable point is the washing of the beads during purification of the cDNA libraries. Cross-contamination of barcode primer plates will also produce multiple clonotypes. In this case, the contaminated P5.IDTxxx.Rd1x.x1 / P7.IDTyyy.Rd2x.x1 barcode primer plate needs to be re-made from stocks.
1Biii or 30	For the bulk RNA protocol, a low number of clonotypes are detected. A possible cutoff is <500 clonotypes (alpha + beta) detected per sample.	This could indicate the fraction of T cells is low in the starting material used to prepare RNA	The read depth can be increased by using a platform other than MiSeq or the amount of input RNA can be increased.
		This could be due to poor RNA quality	Re-extract RNA to obtain a sample with a RIN of at least 4