title: Illumina HiSeq 2500 paired end sequencing; RNA-seq of human cell line HeLa after depletion of a lncRNA with three different loss-of-function methods
design: RNA-seq of human cell line HeLa after depletion of a lncRNA with three different loss-of-function methods
library_construction_protocol: HeLa cells were obtained from American Type Culture Collection. To generate a heterogeneous population of cells expressing dCas9-KRAB, HeLa cells were transduced with a lentiviral vector containing the pHR-SFFV-dCAS9-BFP-KRAB vector along with polybrene (5 ug/ml, Sigma), incubated for 72 hours with medium replacement after 24 hours, and sorted for the BFP-expressing cells using a BD FACSAria III cell sorter. To generate single-cell clones expressing dCas9-KRAB, BFP-sorted single cells were plated on 96-well plates and grown for two weeks until colonies were obtained. Expression of dCas9-KRAB in each colony was verified by Western blot. HeLa cells were maintained in Dulbecco's modified Eagle's medium (Sigma Aldrich, D6429) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and cultured at 37 degrees Celsius with with 5% CO2. For RNAi and LNA, HeLa cells were transfected with Lipofectamine RNAiMax reagent (Thermo Fischer Scientific) following the manufacturer's instructions, using siRNAs (Thermo Fischer Scientifc) and LNA Gapmers (Exiqon) at a final concentration of 50 nM and 25nm, respectively. All experiments were done 48 hr after transfection. For CRISPRi, dCAS9-KRAB transduced cells were plated on a 12-well plate and infected with lentivirus containing the appropriate sgRNA vector. The virus was diluted with HeLa media (1:1 dilution) and cationic polymer polybrene was added to facilitate viral transduction (5 ug/ml, Sigma). After 24 hr incubation, supernatant was removed and fresh media was added for 48 hr before RNA collection. Samples with the same batch number were treated at roughly the same time (within the same month), while the experiment number is nested within batch and refers to cells treated on the same day. The QuantiTect Reverse Transcription Kit (QIAGEN) was used for cDNA synthesis including an additional step to eliminate genomic DNA contamination. RNA (1 ug) was extracted with the RNeasy Kit (QIAGEN) and treated with DNase I following the manufacturer's instructions. RNA quality was assessed using a Total RNA Nano chip with a 2100 Bioanalyzer instrument (Agilent). RNA-seq libraries were prepared from HeLA cells using TruSeq Stranded Total RNA Kit with Ribo-Zero Gold (Illumina, RS-122-2303). Library quality was assessed using a DNA1000 chip with a 2100 Bioanalyzer instrument (Agilent).