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Status |
Public on Nov 15, 2024 |
Title |
RfxCas13d expressing MDA-MB-231 cells, pooled lncRNA library, 14 days after dox induction, reps 1 and 2 |
Sample type |
SRA |
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Source name |
breast
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Organism |
Homo sapiens |
Characteristics |
tissue: breast cell line: MDA-MB-231-Cas13 cell type: cancer cells genotype: lncRNAs knockdown treatment: doxycycline induction
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Treatment protocol |
Cas13d-expressing cells were transduced with the library lentivirus through separate infection replicates by spinfection at 1000 rpm for 1 hour at 37 °C, followed by overnight incubation. After 24 hours, new media with 1 μg/ml puromycin (Invivogen ant-pr-1) was added. Puromycin selection was completed within 48 hours for all cell lines, except for THP1. Because THP1 required an extended selection time, we maintained it in R10 but with 10% Tet-system approved serum (Gibco A4736201) substituted for Serum Plus II and with 1 μg/ml puromycin. THP1 cells took approximately two weeks for full selection. Following puromycin selection, RfxCas13d expression was induced by replenishing the growth medium containing 1 μg/ml puromycin, 5 μg/ml blasticidin and 1 μg/ml doxycycline. Cells were passaged every 2 to 4 days and split as needed, ensuring a guide representation of >1,000×. Samples with a guide abundance of 1,000-fold were harvested at 0, 7, and 14 days post-Cas13d induction.
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Growth protocol |
HEK293FT and MDA-MB-231 cells were cultured in D10 medium: Dulbecco’s Modified Eagle Medium with high glucose and stabilized L-glutamine (Cytiva SH30022.01) supplemented with 10% Serum Plus II (Sigma-Aldrich 14009C) and 5 µg/ml blasticidin. HAP1 and K562 cells were cultured in I10 medium: Iscove’s Modified Dulbecco’s Medium with L-glutamine (Cytiva SH30228.FS) supplemented with 10% Serum Plus II and 5 µg/ml blasticidin. THP-1 cells were cultured in R10 medium: HyClone RPMI 1640 Medium (Cytiva SH30255.FS) supplemented with 10% Serum Plus II and 5 µg/ml blasticidin. All cells were incubated at 37 °C with 5% carbon dioxide.
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Extracted molecule |
genomic DNA |
Extraction protocol |
Genomic DNA was isolated from cell pellets via a modified salting out procedure. For that, 12 ml of NK Lysis Buffer (50 mM Tris, 50 mM EDTA, 1% SDS, pH 8) and 60 µl of 20 mg/ml Proteinase K (QIAGEN 19131) were added to 80 million cells and incubated at 55 °C overnight. The next day, 6 µl of 100 mg/ml RNase A (A.G. Scientific R-2000) was added to the lysed sample, which was then inverted 25 times and incubated at 37 °C for 30 min. Samples were cooled on ice before addition of 4 ml of pre-chilled 7.5M ammonium acetate (Sigma A1542) to precipitate proteins. After adding ammonium acetate, the samples were vortexed and centrifuged at 4,000 × g for 10 minutes. After the spin, 12 ml isopropanol was added to the collected supernatant, inverted 50 times and centrifuged at 4,000 × g for 10 minutes. The supernatant was discarded, 12 ml of freshly prepared 70% ethanol was added, the tube was inverted 10 times, and then centrifuged at 4,000 × g for 10 minutes. The supernatant was discarded, and remaining ethanol was removed. After air drying for 30 minutes, 500 µl of 0.2× TE buffer (Sigma 93283) was added, the tube was incubated at 65 °C for 1 hour and at room temperature overnight to fully resuspend the DNA. The next day, the gDNA samples were vortexed briefly. The gDNA concentration was measured using a Nanodrop (Thermo). We excluded one biological replicate (K562 day 0) due to low recovery after gDNA extraction. We amplified gRNA cassettes and prepared them for sequencing using a two-step PCR. PCR1 was performed to amplify a region containing the crRNA cassette in the lentiviral genomic integrant using TaqB polymerase (Enzymatics P7250L). We performed 70 PCR1 reactions for each gDNA sample using 5 μg gDNA per 100 μl PCR1 reaction as follows: 94 °C for 3 min, 20× (94 °C for 10 s, 55 °C for 30 s, 68 °C for 45 s), 68 °C for 5 min. We then combined PCR1 products for the same sample together before PCR2, which was done to incorporate Illumina adaptors using Q5 High-Fidelity DNA Polymerase (NEB M0491). We performed 10 PCR2 reactions for each sample using 10 μl unpurified PCR1 product per 50 μl reaction as follows: 98 °C for 30 s, 6-10× (98 °C for 10 s, 63 °C for 30 s, 72 °C for 45 s), 72 °C for 5 min. The resulting amplicons from PCR2 (~270 bp) were pooled and then purified using double-sided SPRI beads clean up (Beckman B23317) or gel extracted using a QiaQuick Gel Extraction kit (Qiagen 28704). For double-sided SPRI clean up, the amplicon was first incubated with 0.6× SPRI to remove larger fragments (>350 bp bound to beads and removed), the supernatant was then transferred and further incubated with 0.8× SPRI beads (<200 bp in supernatant and removed).
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Library strategy |
RNA-Seq |
Library source |
genomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
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Description |
MDA-MB-231_Day14_R1, MDA-MB-231_Day14_R2
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Data processing |
Quality of the raw fastq files was assessed using MultiQC .We processed reads from pooled Cas13d screens following established pipelines. In brief, reads were de-multiplexed based on Illumina i7 barcodes and custom i5 barcodes. We trimmed reads to the expected gRNA length by identifying known anchor sequences relative to the guide sequence. We did this using Cutadapt (v.1.13) with the following parameters: -g CTGGTCGGGGTTTGAAAC -e 0.2 -O 5 --discard-untrimmed and -a TTTTTGAATTCGCTAGCT -e 0.1 -O 5 --minimum-length 15 --discard-untrimmed. We aligned processed reads to the designed crRNA reference using bowtie (v.1.1.2) allowing for up to three mismatches (parameters: -v 1 -m 3 –best -q). The raw gRNA counts were normalized using median-of-ratios (geometric mean), similar to DESeq2, and batch correction was applied using combat from the SVA R package (v.3.34.0). We removed nonreproducible technical outliers by pair-wise linear regression for each sample, collecting residuals, and taking the median value for each gRNA across biological replicates. Assembly: GRCh38 Supplementary files format and content: tab-delimited text file include raw counts for each sample.
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Submission date |
Nov 08, 2024 |
Last update date |
Nov 15, 2024 |
Contact name |
Wen-Wei Liang |
E-mail(s) |
[email protected]
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Organization name |
New York Genome Center
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Street address |
101 Avenue of the Americas
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City |
New York |
State/province |
NY |
ZIP/Postal code |
10013 |
Country |
USA |
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Platform ID |
GPL18573 |
Series (1) |
GSE281460 |
Transcriptome-scale RNA-targeting CRISPR screens reveal essential lncRNAs in human cells |
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Relations |
BioSample |
SAMN44614214 |
SRA |
SRX26635954 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
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