- For large cell # (sub 100K – 1000K cells)
- We will be using Zymo’s Direct-zol kits (https://www.zymoresearch.com/direct-zol-isolation-kits) for RNA extraction, unless the clients prefer/provide alternatives. The best way to submit the cells is to ‘lyse’ the cells off from culture plates etc in appropriate amount of TRIzol or TRI reagent (pipetting/vortexing would be required but no further mechanical disruption will be necessary if its from dissociated cells) and either submit the lysate as is or frozen.
- If the cells were frozen by themselves already, NEVER thaw it or NEVER bring the temperature above -80C. Bring the frozen cells out from -80C freezer and immediately place them ON DRY ICE and submit them while keeping them ON DRY ICE. Alternatively, you can add the chilled (kept at 4C) TRIzol or TRI reagent to the frozen cell pellet which is kept ON DRY ICE so that the cells won’t be exposed to elevated temperature without being lysed by TRIzol/TRI reagent, and then immediately vortex or pipet the content to completely lyse the cells. You can submit the cell lysate on ice or at room temperature once the cells are completely lysed.
- For small cell # (1 to a few thousands)
- Spin cells in a wash buffer (e.g. PBS) at moderate speed (e.g. 500Xg, 5 min), using a non-stick RNAse free 0.5ml eppendorf tube. Pipet out supernatant as much as possible by leaving 10-20ul (the less the better). If you can use a swing rotor centrifuge and conical bottom tube, this will be relatively easy but if you have to use an angled rotor, be cautious not disturbing the cell pellet (invisible!) which will be adherent to the outer side of the tube wall, so pipet towards inner side of the tube wall by holding the tube in a way that the inner side will face upside, using a fine-end pipet tip (e.g. 20ul pipet tip). You can spin the tube again if you think you disturbed the cell pellet.
- Add minimum volume of Igepal CA-630 to make final concentration of Igepal at 0.25-1% (Reference for validating optimal cell lysis condition for gene expression study: http://www.nature.com/articles/srep04659) E.g. prepare 20% Igepal (10ml of Igepal + 40ml nuclease free water, mix well, warm the tube if not mixing well), and add 0.5 ul to the 10ul sample with pelleted cells prepared in 1. This will make 1% final Igepal CA-630. The goal here is to minimize the final sample volume (so that most of it can be applied to the library prep: usually 3 ~ 10 ul will be used for the library prep) while ensuring enough concentration of the detergent to lyse the cells. I haven’t validated the inclusion tolerance of higher detergent concentration for the library prep greater than 1% final during the library prep.
- Mix the tube by voltexing 15 sec and snap freeze it by placing onto a crashed dry ice or chilling in a liquid N2.
- Store the tube at -80C. Ship by overnight Fedex/UPS with lots of dry ice. The package sometime delays and excess dry ice will prevent the loss (thawing) of your precious sample! Hershey clients can bring the sample in to the Core using dry ice. Never thaw the sample or let the temperature increase above -80C until the library prep.