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Sample GSM2439131

Query DataSets for GSM2439131

Status

Public on Jan 18, 2017

Title

Bulk_stimulated_RELA_2

Sample type

SRA

Source name

Jurkat cells

Organism

Homo sapiens

Characteristics

cell line: Jurkat
library: RNA-seq
experiment: CROP-seq_Jurkat_TCR
condition: anti-CD3/CD28
knockout: RELA
grna: RELA_2

Treatment protocol

Jurkat cells were serum-starved for 3 hours prior to stimulation. They were stimulated with 1 µg/ml anti-CD3 (eBioscience #16-0037) and 1 µg/ml anti-CD28 antibody (eBioscience #16-0289-81) for 4 hours while under starvation and then subjected to CROP-seq. The unstimulated (naïve) control was subjected to continued starvation.

Growth protocol

Jurkat cells stably expressing SpCas9 (after transduction with lentiCas9-Blast, Addgene #52962) were kept in RPMI medium + 10% FCS + penicillin/streptomycin containing 25 μg/ml blasticidin. After transduction with gRNA-encoding lentiviral particles (CROPseq-Guide-Puro), cells were selected with 2 μg/ml puromycin for 10 days, while keeping up the selection for blasticidin, and then used in T cell receptor stimulation experiments followed by CROP-seq (or bulk RNA-seq on single knockout lines for validation).

Extracted molecule

polyA RNA

Extraction protocol

CROP-seq: Adherent cells were detached using Trypsin-EDTA (Gibco #25300-054), following standard cell culture practices. Cells were collected by centrifugation at 300 rcf for 5 min, washed once with PBS-0.01% BSA (freshly prepared on the day of the run), and resuspended in 1 ml of PBS-0.01% BSA. Cells were filtered through a 40 µm cell strainer to obtain a suspension of single-cells, which were counted using a CASY device. Single cells were then co-encapsulated with barcoded beads (ChemGenes #Macosko-2011-10) using an Aquapel-coated PDMS microfluidic device (FlowJEM), connected to syringe pumps (kdScientific) via polyethylene tubing with an inner diameter of 0.38 mm (Scicominc #BB31695-PE/2). Cells were supplied in PBS-0.01% BSA at a concentration of 220 cells/µl, barcoded beads were resuspended in Drop-seq lysis buffer at a concentration of 150 beads/µl. The flow rates for cells and beads were set to 1.6 ml/hour, while Droplet Generation Oil (BioRad #1864006) was run at 8 ml/hour. During the run, the barcoded bead solution was mixed by magnetic stirring with a mixing disk set to 1 jump/s. A typical run lasted between 35 and 40 min. In case of multiple runs per day, droplets were intermittently stored at 4 °C and processed together. Our most important modification to the protocol is an alternative way to break droplets, which recovers beads much more efficiently than in the original publication of Drop-seq (Macosko et al., 2015). After removing as much oil below the droplet layer as possible, we added 30 ml of 6x SSC buffer (Promega #V4261) and 1 ml of Perfluoroctanol (Sigma Aldrich #370533-25G) and shook the tube forcefully 6 times to break the droplets. Based on their large diameter, beads were then collected by syringe-filtering the solution through a 0.22 µm filter unit (Merck #SLGV033RS), washing 2x with 20 ml of 6x SSC buffer and eluting by turning the filter upside down and rinsing it with 3x 10 ml of 6x SSC buffer. Beads were then collected by centrifugation at 1,250 rcf for 2 min, setting the brake speed to 50%. After washing a second time with 10 ml 6x SSC, the pellet was taken up in 200 µl of 5x RT buffer and transferred to a 1.5 ml tube. Bulk RNA-seq: Total RNA was extracted using the Qiagen AllPrep Kit (Qiagen #80311).
CROP-seq: Reverse transcription and Exonuclease I treatment were performed as described in the original publication, and the number of barcoded beads was estimated using a Fuchs-Rosenthal counting chamber (mixing the bead suspension with 6x DNA loading dye). Depending on the performance of the experiment, we prepared up to 24 PCR reactions per Drop-seq run, adding 4,400 beads (~220 cells) per PCR and enriching the cDNA for 4 + 10 cycles, using the already described reagents. We then prepared Drop-seq libraries using the Nextera XT kit (Illumina #15032350), starting from 1 ng of cDNA pooled in equal amounts from all PCR reactions for a given run. We typically required an additional 10 enrichment cycles, using the Illumina Nextera XT i7 primers along with the Drop-seq New-P5 SMART-PCR hybrid oligo. The slightly increased cDNA input typically results in an average size distribution of about 575 bp. After quality control, libraries were sequenced with paired-end SBS chemistry on Illumina HiSeq 3000/4000 instruments. Drop-seq Custom Read1 Primer was spiked into the HP10 primer solution, located in column 11 of the cBot Reagent Plate at 1µM final concentration. High sequence complexity needed for optimal base calling performance was achieved by adding 20-30% PhiX as spike-in. Cluster generation and Read 1 primer hybridization were completed using cBot protocol ‘HiSeq_3000_4000_HD_Exclusion_Amp_v1.0’. Alternatively, libraries were sequenced on an Illumina NextSeq 550 instrument using the 75 cycle High Output Kit. We loaded 1.8 pM library and provided Drop-seq Custom Read1 Primer at 0.3 µM in position 7 of the reagent cartridge. On NextSeq machines, we sequenced without PhiX spike-in, using a read configuration of 20 bases (Read1), 8 bases (Index) and 64 bases (Read2); Bulk RNA-seq: Bulk RNA-seq libraries were prepared using the QuantSeq 3' mRNA-Seq Library Prep Kit (Lexogen #015.96) according to the manufacturer's instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

RNA-seq on a bulk population of Jurkat cells treated with anti-CD3/CD28 antibodies and transduced with gRNA RELA_2
processed data file: CROP-seq_Jurkat_TCR.count_matrix.csv.gz

Data processing

Single-cell sequencing: Data were processed using the Drop-seq Tools v1.12 software (Macosko et al, 2015). Briefly, each transcriptome Read 2 was tagged with the cell barcode (bases 1 to 12) and UMI (unique molecular identifier) barcode (bases 13 to 20) obtained from Read 1, trimmed for sequencing adapters and poly-A sequences, and aligned using STAR v2.4.0 15 to the human reference genome assembly (Ensembl GRCm38/GRCh38 release) containing artificial chromosomes that represent the CROPseq-Guide-Puro plasmid construct (250 bp common U6 promoter sequence, one gRNA sequence per artificial chromosome (20 bp), and the remaining 260 bp downstream plasmid backbone until the poly-A sequence). Cell barcodes were corrected for possible bead synthesis errors, allowing the removal of up to 4 bases using the DetectBeadSynthesisErrors tool from the Drop-seq Tools v1.12 software. Reads aligning to exons were tagged with the respective gene name, and counts of unique UMIs per gene within each cell were used to build a digital gene expression matrix for cells with counts for at least 500 genes. This matrix was converted to transcripts per million and log-transformed for further analysis. We excluded genes coding from ribosomal proteins from the analysis as we suspect their widespread detection is caused by solubilization of their mRNAs as noted previously (Macosko et al, 2015). For the assignment of gRNAs to cells, we quantified the overlap of reads to the specific gRNA sequence within the CROPseq-Guide-Puro plasmid chromosomes and assigned the most abundant gRNA to the respective cell.
Bulk RNA sequencing data: Reads were trimmed with Trimmomatic and aligned to the GRCh38 assembly of the human genome using Bowtie1 with the following parameters: -q -p 6 -a -m 100 -minins 0 maxins 5000 –fr –sam -chunkmbs 200. Duplicate reads were removed with Picard’s MarkDuplicates utility with standard parameters before transcript quantification with BitSeq 18 using the Markov chain Monte Carlo method and standard parameters. To obtain gene-level quantifications, we assigned the expression values of its highest expressed transcript to each gene.
Genome_build: hg38
Supplementary_files_format_and_content: Comma-separated values (CSV) files with genes as rows and samples/cells as columns. For the file with the single-cell expression values, we added 4 metadata header rows (stimulation condition, microfluidics run ("replicate"), cell barcode, assigned gRNA, targeted gene), which refer to the respective cells. The "replicate" field indicates the run referenced in the sample title.

Submission date

Dec 23, 2016

Last update date

May 15, 2019

Contact name

Christoph Bock

E-mail(s)

cbock@cemm.oeaw.ac.at

Organization name

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences

Street address

Lazarettgasse 14