title: Tbx21_3_7_11
organism: Mus musculus
description: Protocols: 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.
attributes.tag: ENA first public
attributes.value: 2017-12-12
attributes.tag: ENA last update
attributes.value: 2017-12-12
attributes.tag: External Id
attributes.value: SAMEA104424244
attributes.tag: INSDC center alias
attributes.value: Sanger Institute / Karolinska Institutet
attributes.tag: INSDC center name
attributes.value: Sanger Institute / Karolinska Institutet
attributes.tag: INSDC first public
attributes.value: 2017-12-12T17:01:54Z
attributes.tag: INSDC last update
attributes.value: 2017-12-12T14:57:09Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6285:Tbx21_3_7_11
attributes.tag: age
attributes.value: 6 to 8
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: T-helper 2 cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: genotype
attributes.value: Rosa26-Cas9; Tbx21-targeting sgRNA 2
attributes.tag: individual
attributes.value: 3
attributes.tag: organism part
attributes.value: spleen
attributes.tag: phenotype
attributes.value: CRISPR/cas9 mediated knockout of Tbx21
attributes.tag: sample name
attributes.value: E-MTAB-6285:Tbx21_3_7_11
attributes.tag: strain
attributes.value: mixed