Illumina HiSeq 2500 paired end sequencing; GSM1640496: 0610009D07Rik No LPS (0610009D07Rik..MGLibA_00009snnT0); Mus musculus; RNA-Seq

0610009D07Rik No LPS (0610009D07Rik..MGLibA_00009snnT0)

We introduced genome-wide pooled CRISPR-Cas9 libraries into primary mouse dendritic cells (DCs) to identify genes that control the induction of tumor necrosis factor (TNF) by bacterial lipopolysaccharide (LPS), a key process in the host response to pathogens, mediated by the TLR4 pathway. We found many of the known regulators of TLR4 signaling, as well as dozens of previously unknown candidates that we validated. Overall design: We used stain base phenotype (staining for TNF) in order to search for negative and positive regulators of LPS response in differentiated BMDCs

A genome-wide CRISPR screen in primary immune cells to dissect regulatory networks

Submitted by Gene Expression Omnibus on 17-JUL-2015

RNA was purified using Qiagen RNAeasy 96 Kit according to the manufacturer’s instructions. The RNA was eluted in a volume of 50ml. For library construction we used the SMART-seq2 protocol (Picelli et al., 2013) in a 96 well plate format and with several modifications. 2 μl of RNA sample per well were mixed with 2ml RT primer (10mM 5 ́-AAGCAGTGGTATCAACGCAGAGTACT30VN-3 ́), 2ml dNTP mix (10mM each, Agilent Technologies) and 2ml Recombinant RNase Inhibitor (RRI-Clontech). This mix was incubated for 3min at 72°C and immediately placed on ice. To perform reverse transcription (RT) we added a mix of 1.5ml H2O, 4 ml Maxima buffer (ThermoFisher Scientific), 4 ml Betaine (5M SIGMA-ALDRICH), 1.8 ml MgCL2, 2ml TSO (10mM AAGCAGTGGTATCAACGCAGAGTACrGrG+G), 0.5 RRI and 0.2ml Maxima H Minus Reverse Transcriptase enzyme (ThermoFisher Scientific). We incubated the RT reaction mix at 42°C for 90 min followed by 10 cycles of 50°C for 2 min, 42°C for 2 min, afterwards heat inactivated the enzyme for 15 min at 70°C. We used 11ml of the RT reaction for the PCR reaction by adding 12.5 ml KAPA HiFi Hotstart (KAPA Biosystems), 1ml H2O and 0.5 ml of (10mM 5’-AAGCAGTGGTATCAACGCAGAGT-3 ́) primer under the following conditions: 98°C for 3 min, 14 cycles of (98°C for 15 sec, 67°C for 20 sec, 72°C for 6 min), final extension at 72°C for 5 min. The PCR product was used for library preparation with Nextera XT DNA Sample Preparation (Illumina) according to the manufacturer’s instructions. Samples were combined and purified using Ampure XP Agencourt beads (Beckman Coulter) and sequenced on a Hi-Seq 2500 (Illumina), to generate paired-end 25bp reads. Each sample was sequenced to an average depth of four million reads (IQR-2.3 -5.5 million).

A genome-wide CRISPR screen in primary immune cells to dissect regulatory networks

Submitter Supplied Information

Samples