GSM3301591: Nr5a2-/- clone #130 D6 PGCLC; Mus musculus; RNA-Seq
For fluorescence-activated cell sorting (FACS), cultured cells or embryoids were dissociated into single cells with TrypLE, and suspended in phosphate buffered saline (PBS) with 0.5% BSA or 3% FBS. Filtered cell suspensions were subsequently sorted using a Sony SH800Z or a MoFlow high-speed cell sorter (Beckman Coulter) or analysed with BD LSRFortessa X-20 (BD Biosciences). For exit from pluripotency analyses, cells were sorted/analysed based on absence or presence of Stella-GFP expression, whilst maintaining Esg1-tdTomato activitaion (SG+/-ET+). For PGCLC, cells were sorted/analysed based on Stella-GFP re-activation coupled with low Esg1-tdTomato. The threshold(s) levels for sorting was set and normalised based on expression levels in pre-optimised SGET ESC. A negative population of ESC without any fluorescence was used to set absolute thresholds. Forward and side scatters were used to gate for the cell population and doublets. For each sort the maximum number of cells was collected. Sample1-36: For RNA-seq of Nr5a2-/-, Zfp296-/- and matched WT control cell lines, 100 ng of total RNA from triplicate biological independent experiments was used as inputs for the NEBNext Ultra RNA library Prep Kit for Illumina (NEB), with the library generated as per manufacturer's instructions. During the final PCR amplification stage, 15 cycles of amplifications were performed to generate the adaptor ligated, fragmented cDNA for sequencing. Samples were assessed using the High Sensitivity D1000 ScreenTape assay (Agilent) to ensure the library did not contain primer-dimer contamination after last round of AMPure beads cleaning. The Qubit dsDNA HS assay kit (ThermoFisher Scientific) and the NEBNext Library Quant Kit for Illumina was used to accurately quantify the concentration of each library preparation. Samples were multiplexed with 12 indexes per lane and a total of 2 lanes of sequencing on Illumina HiSeq4000, single-end 50bp with an average depth of approximately 20 million reads per sample. Sample 37-42: ESC, EpiLC, or day 6 PGCLC (from the CRISPR screen) were dissociated into single cell solutions with TrypLE and sorted with a Sony SH800Z or a MoFlow high-speed cell sorter (Beckman Coulter) based on appropriate Stella-GFP and Esg1-tdTomato expression. Samples were sorted into 150ul of extraction buffer from the PicoPure RNA isolation kit (Life Technologies) and rapidly frozen on dry ice or liquid nitrogen. Total RNA was subsequently extracted using the PicoPure RNA isolation Kit, including a 15 minute on-column DNaseI digestion. RNA integrity number was assessed with RNA HS ScreenTape (Agilent), and all samples confirmed to have a RIN >8.5. For RNA-seq from CRISPR screen samples, 50 ng of total RNA was used as input for the Ovation RNA-seq System v2 (Nugen) as per manufacturer's instructions. Amplified double-stranded (ds) cDNA was diluted into EDTA-low TE (Agilent) and sheared into ~230bp in length using S220 Focused-Ultrasonicator (Covaris) using settings: duty factor 10%, cycle burst 200, intensity 5, temp at 4°C and treatment time of 5 minutes per sample.  A high Sensitivity D1000 ScreenTape assay (Agilent) was used to assess the efficiency of library fragmentation. Fragmented ds-cDNA was concentrated with Qiagen Reaction Clean Up kit (MiniElute) and 1μg of the fragmented ds-cDNA used as input for the library preparation using Encore Rapid DR Multiplex Library System (Nugen). This kit ligated the adaptors to repaired-end ds-cDNA without amplification, which eliminated biases introduced during PCR amplification. The KAPA Library Quantification Kit (Kapa bioscience) was used to quantify the concentration of each adaptor-ligated libraries prior to multiplexing. The ESC, EpiLC and PGCLC RNA sequencing libraries were generated in parallel for each replicate, and the biological replicates were generated on independent occasions. Samples were multiplexed and sequenced with Illumina HiSeq1500, single-end 50bp read length with a minimum depth of approximately 15 million reads per sample.  Sample43-52: To quantify the relative frequencies of integrated gRNAs during the CRISPR screen in each population during cell fate transitions, genomic DNA was isolated from day 12 ESC post-lentiviral transduction, from SG–ET+ and SG+ET+ EpiLC populations at 42hr, and from SG+ETlow PGCLCs from day 6 embryoids, in biological duplicate using the DNeasy Blood & Tissue kit (Qiagen). Purified genomic DNA was used as template for custom primers that specifically amplify the gRNA region, and include overhanging Illumina adaptors and indexes to allow deep sequencing and multiplexing, respectively (synthesised as Ultramers from IDT) (see oligo tables). Prior to PCR, genomic DNA was sonicated with a Biorupter (Diagenode) for 15 seconds on 'LOW' power to improve efficiency of amplification. gRNA sequences were amplified in multiple PCR reactions to enable all isolated DNA to be utilised, each with NEBNext Q5 HotStart HiFi PCR Master Mix (NEB), 0.2 uM of universal forward primer (mix of staggers), 0.2 uM of indexed reverse primer and genomic DNA (~650 ng / 50 ul rxn). For amplification from the plasmid vector, 15 ng was used as template for replicate amplifications. The following cycling conditions were used: 95°C for 2 minutes, then between 21-26 cycles of (98°C for 20 seconds, 62°C for 20 seconds and 72°C for 20 seconds) followed by 72°C for 1 minute and hold at 4°C. The number of cycles was optimised for the point at which PCR products can be first visualised on an agarose gel. The PCR reaction was subsequently purified with AMPure XP beads (Beckman Coulter) using double size selection to remove primer dimers and genomic DNA. Briefly, 0.55x of AMPure beads were added to PCR reaction (1x) and incubated for 5 minutes at room temperature. The supernatant was collected; the beads contained the unwanted larger fragments and were discarded. An additional 0.3x of AMPure beads was subsequently added to the supernatant (1x) and incubated for 5 minutes at room temperature. The supernatant was removed and beads washed twice with 200 ul of 80% EtOH, air dried for 5 minutes and DNA was eluted from AMPure beads with EDTA-low TE buffer. The concentration of adaptor ligated gRNA amplicons was measured with the Qubit DNA Assay kit (ThermoFisher Scientific) and the fragment distribution determined with an Agilent D1000 ScreenTape System (Agilent Technologies). Libraries were subsequently multiplexed and sequenced with an Illumina HiSeq 1500 using single-end 50bp reads. As the sequences for all samples were identical up to the gRNA region, these types of low complexity libraries can produce low quality data. To counteract this, forward staggered primers were introduced at equal ratios during PCR amplification, generating offset reads. In addition, libraries were sequenced with 4 dark cycles and low density (70%) clustering. Sample 1-42: RNA-seq; Sample 43-52: amplicon