Problem	Possible reason	Solution
Cell not transferred into lysis tube	Cell stuck to the inside wall of the glass capillary (micropipette)	First suck a small volume of PBS-BSA using the mouse pipette, then suck the single cell into the glass capillary gently to keep the cell already inside the capillary but still near the tip of the capillary. When pushing the cell out of the capillary into the lysis, push all the carryover PBS-BSA until a bubble is visible from the capillary into the lysis buffer.
Picking buffer only control shows positive signal when using real-time PCR to check expression of house-keeping genes	PBS-BSA drop is contaminated by lysed cells	Make sure the PBS-BSA drop for holding single cells is not contaminated by lysed cells. Wash the single cells through several PBS-BSA drops before picking them. Aliquot all reagents for lysis, RT, cutting, tailing, and PCR steps in small batches. Each aliquot is only used once and the remainder is discarded. Also only load the exact number of single cells that will be picked into the final PBS-BSA drop. After picking each of them, check the number of remaining single cells to make sure every cell is correctly picked and no cell is by chance lysed or incorrectly picked.
The expression level of house-keeping genes is much lower than expected in some single cell cDNA samples.	Some dead or partially damaged single cells were picked	Before picking, try to check the quality of the single cells by either morphology or trypan blue staining. Only pick healthy single cells.
	RNase contamination during the lysate and reverse transcription step	Keep the bench and surrounding area very clean. Wear a dusk mask to avoid breathing contaminates into reaction reagents. Change gloves regularly during these steps. Try to do all manipulations in a clean hood.
	Loss of activity of some enzymes and reagents	Some of the reagents are not very stable, such as dATP, dNTP, or primers at low concentrations. Aliquot them into small batches. Avoid repeated freeze/thaws. Make sure reagents are not expired.
Low recovery of gel purification	Gel fragment recovered more than the kit requested	Try to cut the gel fragment containing the cDNA smear more accurately. If too much gel is already cut, split them into two gel purification columns and finally combine the purified cDNAs. Each QIAGEN column can hold up to 400 mg of gel.
Primer dimers present on Lonza Gel in step 70	Too many cycles of PCR were done, or the amount of adaptors added was too large relative to sample size	Gel purification must be done to remove primer dimers. SOLiD Fragment Library protocol recommends the use of 3% or 4% agarose gel run in 1X TAE, using a 25 bp Track-It ladder as reference. Cut the gel between 125 bp and 200 bp and purify using QIAGEN MinElute Gel Extraction Kit following manufacturer’s instructions.
Size distribution of fragment after shearing is not in the desired range	Sonication protocol was not calibrated correctly for the sample	Readjust shearing protocol and redo shearing. Make sure the Covaris S2 System is properly set up.
Low yield after AMPure purification	Ethanol was not freshly prepared, and/or was not at 70%	If there is adequate sample remaining, PCR can be done to increase the quantity, see steps 69-72. If not, protocol must be redone, using freshly prepared 70% ethanol.
Overall yield is low	Sample quality was poor	Confirm quality of sample before beginning Express protocol using a housekeeping gene assay, or use Qubit to confirm that the sample’s concentration is adequate for input.
	Sample loss occurred during AMPure purification	See above. Check QC aliquots using Qubit or NanoDrop to determine where loss occurred.
	Loss of sample can occur during steps when sample is transferred or purified	Take care to transfer the entire volume during purification steps and minimize transferring of samples. Sample input may need to be increased if initial input was low and loss occurs.