|Public on Apr 13, 2017
|K562 dCas9-KRAB, Lenti-sgRNA44_bc15
|cell line: K562
|HEK293T cells were cultured in DMEM with 10% FBS and pen/strep. For lentiviral packaging, 3X106 293T cells were seeded in a 6 cm dish one day before transfection. The indicated viral plasmid(s) were co-transfected with lentivirus packaging plasmids pMD2.G and psPAX2 (Addgene ID 12259 and 12260) with 4:2:3 ratio by using Lipofectamine 3000 (Thermo Fisher) according to the manufacturer's protocol. Twelve hours after transfection, media was changed to fresh DMEM with 10% FBS plus Pen/Strep. Seventy-two hours after transfection, virus-containing media was collected, passed through a 45 um filter, and aliquoted into 1.5ml tubes. Viruses were stored in -80Â°C before infection or titration. For virus infection, 2X105 K562 cells were seeded in 24 well plate per well with 500 Âµl complete medium plus 1Âµg/ml polybrene. Virus aliquot(s) were thawed to room temperature and added to the plates. The plate was then centrifuged at 1000xg at 36 Â°C and returned to the incubator. The following day, the media was changed to fresh complete medium with antibiotics to screen for infected cells. Cells were kept at ~30% confluence during antibiotic selection.
|K562 were cultured in IMDM plus 10% FBS and pen/strep at 37C and 5% CO2.
|total RNA was extracted from cells using Trizol Reagent (Thermo), and were subjected to enrichment of polyA+ RNA using the NEBNext Poly(A) kit (NEB).
Strand-specific polyA+ RNA-Seq was performed using the LM-Seq method, as described previously. Briefly, PolyA+ RNA was fragmented at 85Â°C in SmartScribe first-strand buffer (CloneTech) supplemented with a 3â sequencing adapter containing a random hexamer sequence. The fragmented RNA were subjected to first strand cDNA synthesis, followed by RNase A/H treatment. The remaining single-stranded DNA were ligated to a 5â sequencing adapter. The libraries were then amplified by PCR using Illumina TruSeq-compatible primers and purified using SPRI purification beads.
|Illumina NextSeq 500
|sgrna barcode id: bc15
sgrna id: sgRNA44
sgrna sequence: ggactatgggaggtcactaa
sgrna target: beta globin HS2
|Basecalls performed bcl2fastq v188.8.131.52.
RNA-seq reads were aligned using the STAR aligner v2.4.2a with default parameters to the hg19 genome assembly augmented with a sequence representing the sgRNA-containing plasmid with a fully degenerate 12-bp barcode sequence.
10, and PCR duplicates were removed with Picard v1.117.
Read counts were tabulated with featureCounts v1.5.0.
Supplementary_files_format_and_content: read counts for all genes
|May 13, 2016
|Last update date
|May 15, 2019
|Gary Chung Hon
|5323 Harry Hines Blvd.
|Multiplexed engineering and analysis of endogenous enhancer activity in single cells: Bulk RNA-Seq validation
|Multiplexed engineering and analysis of endogenous enhancer activity in single cells