title: GSM5825542: CRISPRaX_Screen_Unsorted_r1; Mus musculus; OTHER
library_construction_protocol: The PrimeFlow RNA assay (Thermofisher) was used according to the manufacturer's recommendations. Specifically, the assay was performed in conical 96-well plates for a total of 200 million cells per replicate with 5 million cells per well  using Xist specific probes, labelled with Alexa-Fluor647 (VB1-14258). 20 million cells were snap-frozen after the two fixation steps to be used as the Unsorted fraction.  Samples were resuspended in PrimeFlow™ RNA Storage Buffer before flow cytometry. Here, four different populations were sorted according to Xist expression. In more detail, a Xist-negative population was taken from the bottom-15% of Xist-expressing cells. Three Xist-positive populations were taken in 15%-intervals from the top Xist-expressing cells (0-15% = High, 15-30% = Medium, 30-45% = Low). Around 11-15 million cells were recovered per fraction. After sorting, the cell pellets were snap-frozen and stored at -80°C.  DNA from frozen cell pellets was isolated through phenol/chloroform extraction. Cell pellets were thawed and resuspended in 250 µl of lysis buffer (1% SDS (Thermo Fisher Scientific), 0.2 M NaCl and 5 mM DTT (Roth) in TE Buffer) and incubated overnight at 65°C. The next day 200 µg of RNAse A (Thermo Fisher Scientific) were added and the samples were incubated at 37°C for 1 h. 100 µg of Proteinase K (Sigma) were subsequently added, followed by a 1 h incubation at 50°C. Phenol/chloroform/isoamyl alcohol (Roth) was added to each sample in a 1:1 ratio, the mixture was vortexed for 1 min and subsequently centrifuged at 16,000 x g for 10 min at room temperature. The aqueous phase was transferred to a new tube, 1 ml 100% EtOH, 90 µl 5 M NaCl and 1µl Pellet Paint (Merck) was added to each sample, mixed, and incubated at -80°C for 1 h. DNA was pelleted by centrifugation for 16,000 x g for 15 min at 4°C, pellets were washed twice with 70% EtOH, air-dried and resuspended in 50 µl water. Sequencing libraries were prepared from both sorted and unsorted cell populations for each of the three independent screen replicates. Genomic DNA from frozen cell pellets was isolated by Phenol/Chloroform extraction. Briefly, cell pellets were thawed and resuspended in 250 µl of Lysis buffer (1% SDS (Thermo Fisher Scientific), 0.2 M NaCl and 5 mM DTT (Roth) in TE Buffer) and incubated overnight at 65 °C. 200 µg of RNAse A (Thermo Fisher Scientific) were added to the sample and incubated at 37 °C for 1 h. 100 µg of Proteinase K (Sigma) were subsequently added followed by a 1 h incubation at 50 °C. Phenol/Chloroform/Isoamyl alcohol (Roth) was added to each sample in a 1:1 ratio, the mixture was vortexed at room temperature for 1 min and subsequently centrifuged at 16,000 x g for 10 min at room temperature. The aqueous phase was transferred to a new tube, 1 ml 100% EtOH, 90 µl 5 M NaCl and 2 µl Pellet Paint (Merck) was added to each sample, mixed, and incubated at -80 °C for 1 h. DNA was pelleted by centrifugation for 16,000 x g for 15 min at 4 °C, pellets were washed twice with 70% EtOH, air-dried and resuspended in 50 µl H2O.  The genomically integrated sgRNA cassette was PCR-amplified to attach sequencing adaptors and sample barcodes. To ensure proper library coverage (300x), a total of 20 µg of each sample were amplified using the ReadyMix Kapa polymerase (Roche) with a total of 25 cycles and an annealing temperature of 56 °C. A relatively low amount of 0.5 µg genomic DNA was amplified per 50 µl PCR reaction since in samples stained with Flow-FISH, PCR amplification was inhibited at higher DNA concentrations. PCR was performed with the primer LR175 in combination with a sample-specific primer which contains a distinct 6-nucleotide barcode to allow sample identification after multiplexed deep sequencing (Primer sequences in Suppl. Table 4, LR178/LR180). Successful amplification was verified on a 1% agarose gel and the reactions were pooled. 1 ml of each pooled PCR was purified using the QIAquick PCR Purification Kit (Qiagen) and the resulting amplicons were loaded on a 1% agarose gel and purified using the QIAquick Gel Extraction Kit (Qiagen). CRISPR screen