Cells were xed with PFA for min at room temperature.Cells were washed with PBS for three times at room temperature.Cells were washed for another three times in PBS and covered in ul PBS.Plates were imaged using IXU ultra platescanning confocal microscope at X magnication.For each well of well plate, both OCT immunouorescence and DAPI image were taken in the elds of the well to represent each well.The average OCT immunouorescence was normalized using the regression formula.Zscore was calculated as previously described swas used.A cutoff at was used to identify hits from the CRISPR screen.Then cells were counted and K cells were seeded into one well of well plates for each binding site KO.The cells were harvested hours after transfection of the sgRNA constructs and a cell count was performed using a haemocytometer.The cells were spun down at rpm for minutes at room temperature.Following this, the cells were resuspended in mESC media without LIF in order to allow subsequent differentiation of the cells.The density of the cell suspension after resuspension was xed at cellsmL.For each individual treated and control cell suspension, uL droplets of the suspension were deposited on the lids of cm sterile culture plates.A total of droplets were deposited on each lid to initiate EB formation via the hanging drop method. The culture plate itself was lled with mL of phosphate buffered saline buffer to ood the lid.CO for hours till observable suspended embryoid bodies can be seen.Cells were harvested with passive lysis buffer hours posttransfection.The reprogramming medium was changed every day.The transfection mix was added into each well of well plate with cells.Triton X for min, and blocked with FBS in PBS for min at room temperature.In brief, mRNA was puried from total RNA by incubating with polyT oligoattached magnetic beads.Following purication, mRNA was fragmented into small pieces using divalent cations at an elevated temperature.The cleaved RNA fragments were reverse transcribed into rststrand cDNA using reverse transcriptase and random primers.These strandspecic cDNA fragments undergo an end repair process to produce a buy Sulforaphene single A base overhang, followed by ligation of the adapters.The products were puried and enriched with PCR to create the nal cDNA library.The blot was subsequently incubated with horseradish peroxidase. Three or less mismatches were allowed in each read.The mapped libraries were then subjected to cuffdiff to identify the differentially expressed genes and FPKM values of genes in different samples.The matrix was uploaded to R and normalized perrow using the following formula. A cutoff at was used to identify hits from the FOCUS screen.All the information regarding the statistical analysis, sample size, applied statistical tests, and signicance is reported in the gures and corresponding gure legends.Differences between two groups or multiple groups for the qRTPCR data were analyzed by the Studentsttest with apvalue less than. For qRTPCR, replicates are considered as the RNA samples treated with the same condition within one experiment setup. e Cell Reports, October, As a major hormone of the pineal gland, melatonin exerts many beneficial effects on TBI, but there is no information regarding the effects of melatonin on ferroptosis after TBI.