Illumina HiSeq 2500 paired end sequencing; RNA sequencing of T helper cell type 2, with CRISPR KO
RNA sequencing of T helper cell type 2, with CRISPR KO
Naive T cells were extracted from 3 mice independently, with the Naive CD4+ T Cell Isolation Kit (Miltenyii #130-104-453) according to manufacturer's instruction. Cells were seeded at 200k/well density in 96w format. On day 0, the cells were then resuspended in warm IMDM supplemented with 2-Mercaptoethanol “BME” (50 uM Gibco #31350010), IL4 (10ng/ml, R&D Systems 404-ML), IL2 (6ng/ml) and anti-CD28 (3ug/ml, eBioscience #16-0281-86) and Pen/Strep, before being seeded onto the 6-well plates (30-40M cells per plate). On day 1, the cells were infected by the following procedure. A virus containing was added to each well. This media consisted of 80% virus, 20% IMDM, supplemented with BME/IL2/IL4/anti-CD28 at concentrations as before. In addition, the media contained 8ug/ml polybrene. The plate was put in a zip-lock lag and spun at 1100g for about 2 hours at 32°C. The plate was then put in an incubator over-night (never more than 24h in total). The cells in the media were spun down (the cells attached kept in place) and resuspended with media as after the T cell extraction except with the addition of 2ug/ml puromycin. On day 5, cells were washed and dead cells removed by low-G spin. This typically raised the viability from roughly 10-20% to 60% according to Trypan blue. Cells were spun down and as much of the media removed as possible. Up to 100ul of buffer RLT+ was then added to each well and plates frozen. Later, plates were thawed and RNA extracted by adding 100ul of Ampure XP beads. Cells were lysed with RLT+ and RNA purified using ampure XP beads RNA purification was done by a robot, with 2x200ul EtOH wash and final suspension in RNAse-free water. RNA was then diluted to 500ng/ul and 2ul was taken as input into non-capping DogSeq (manuscript in preparation). This protocol is for this application roughly equivalent to Smartseq-280. Libraries were made using Nextera XT.
Experimental Factor: genotype
Rosa26-Cas9; Thy1-targeting sgRNA 1
Experimental Factor: phenotype
CRISPR/cas9 mediated knockout of Thy1
Experimental Factor: block
3