after washing, u6 probe hybri diz ation was performed following the same protocol as for cells. after hybri diz ation, tissue sections were washed with 0. 1 % trit on x - 100 in pbs. blocking was then performed using 3 % chicken egg white albumin ( sigma - aldrich, a5503 ) in pbs for 1 h at rt. primary antibod ies were applied, followed by incubation with the corresponding alexa fluor - conjugated secondary antibod ies. tissue samples were then fixed in prolong gold antifade with dapi ( invitrogen, p36931 ). cells and tissue samples were then imaged with the sora marianas spinning disk confocal microscope ( intelligent imaging innovations, 3i ) and a fluorescence microscope ( olympus ix71, olympus life science, japan ), respectively. cells were washed twice with ice - cold pbs and lysed with cell lysis buffer ( cell signaling, # 9803 ) or ripa buffer ( cell signaling, # 9806 ) containing inhibitors for proteases and phosphatases. cell lysates were resolved by sds - page and transferred onto nitrocellulose membranes followed by blotting. the information for the antibod ies for immunoblotting is shown in supplementary table s5. single cell suspensions were prepared from spleen and skin tissues. live / dead labeling was performed before the cell surface staining, using a zombie nir™ fixable viability kit ( biolegend; catalog number 423106 ) diluted 1: 1, 000 in pbs for 10 min at room temperature in dark. cells were labeled with specific fluorescence - conjugated antibod ies to cd45, cd3, cd4, cd8, mhcii, cd11b, cd103, ly6c, and / or ly6g for 20 min on ice. intracellular cytokine staining was performed using an ebioscience intracellular fixation & permeabilization buffer set ( catalog number 88 - 8824 - 00 ) according to the manufacturer ’ s instructions. antibod ies against foxp3 ( clone; nrrf - 30 ) were used for regulatory t cell staining. flow cytometric analysis was performed on a fortessa 4 – 15 ( bd biosciences ) and attune nxt ( thermo fisher scientific ) with flowjo v10. 6. 1 used for analysis. the information for the antibod ies is shown in supplementary table s6. the gating strategy is shown in supplementary fig. s18. 100 μg to 150 μg total rna was extracted from cells using trizol following the manufacturer ’ s protocol. mrna was purified using a dynabeads mrna direct kit following the manufacturer ’ s protocols. 1 μg mrna was sonicate d to ∼100 nt and ma containing mrna fragments was enriched with an epimark n6 - methyladenosine enrichment kit following the manufacturer ’ s protocols. finally, rna was isolated using rna clean and concentrator ( zymo research ) and subjected to library preparation with a truseq stranded mrna sample preparation kit ( illumina ). the ma enrichment in mrna and u6 snrna was analyzed using ultra – high - performance liquid chromatography - tandem mass spectrometry ( uhplc - ms / ms ). total rna was extracted from cells using trizol following the manufacturer ’ s protocol. u6 snrna was isolated as described previously. the ma in polyadenylated rna was quantified using an agilent 6460 lc - ms / ms spectrometer as described previously general pre - processing of reads: all samples were sequenced by illumina hiseq4000 with single end 80 bp read length. the adapters were removed by using cutadapt for ma - seq. refseq gene structure annotations were downloaded from the ucsc table browser. reads were aligned to the reference genome ( hg38 ) using hisat2. deseq2 was used for differential gene expression analysis. lowly expressed genes were pre - filtered out using reads count threshold of 10. an adjusted p value ( deseq2 default ) threshold of 0. 05 was used for all differential expression analysis. aligned reads were extended to 150 bp ( average fragment size ) and converted from genome - based coordinates to isoform - based coordinates. the method used for peak calling was adapted from published work with modifications. to call ma peaks, the longest isoform of each gene was scanned using a 100 bp sliding window with a 10 bp step. to reduce bias from potential inac curate gene structure annotation and the arbitrary usage of the longest isoform, windows with read counts of less than 1 / 20 of the top window in both ma - ip and the input sample were excluded. for each gene, the read counts in each window were normalized to the median count of all windows of that gene. a fisher exact test was used to identify the differential windows between ip and input samples. the window was called as positive if the fdr < 0. 01 and log2 ( enrichment score ) > = 1. overlapping positive windows were merged. the following four numbers were calculated to obtain the enrichment score of each peak ( or window ): read counts of the ip samples in the current peak / window ( a ), median read counts of the ip sample in all 100 bp windows on the current mrna ( b ), read counts of the input sample in the current peak / window ( c ), and median read counts of the input sample in all 100 bp windows on the current mrna ( d ). the enrichment score of each window was calculated as ( a×d ) / ( b×c ). the data have been deposited in the geo repository with accession number gse145925. real - time quantitative pcr ( qpcr ) was performed to assess the relative ma abundance of the selected u6 snrna and mrna in ma antibod y ip samples and input samples, as described previously. briefly, total rna was isolated with an rneasy mini kit or trizol. while 500 ng rna was saved as an input sample, the rest of the rna was used for ma ip. 100 μg rna was diluted into 500 μl ip buffer ( 150 mm nacl, 0. 1 % np - 40, 10 mm tris, ph 7. 4, 100 u rnase inhibitor ) and incubated with the ma antibod y ( synaptic systems, goettingen, germany ). the mixture was rotated at 4 °c for 2 hours, then dynabeads® protein a ( thermo fisher scientific, waltham, ma ) coated with bsa was added into the solution and rotated for an additional 2 hours at 4 °c. after washing with the ip buffer with rnase inhibitors four times, the ma ip portion was eluted with elution buffer ( 5 mm tris-hcl ph 7. 5, 1 mm edta ph 8. 0, 0. 05 % sds, and 4. 2 μl proteinase k ( 20 mg / ml ). the final eluted rna was concentrated with an rna clean & concentrator - 5 kit ( zymo research, irvine, ca ). the same amount of the concentrated ip rna or input rna from each sample was used for the cdna library. the rna expression was determined by the number of amplification cycles ( cq ). the relative ma levels in genes were calculated by normalizing the ma levels ( ma ip ) against the expression of each gene ( input ). endo - ip was performed as described previously. cells were harvested on ice by scraping in 2 ml dpbs and pelleted by centrifugation at 1000 x g for 2 min at 4 °c. supernatant was discarded, and cell pellet was washed once with 1 ml of kpbs buffer ( 25 mm kcl, 100 mm potassium phosphate, ph 7. 2 ), followed by centrifugation at 1000xg for 2 min at 4 °c. cells were then resuspended in 500 µl of kpbs supplemented with the protease inhibitors ( thermo fisher, 78442 ) and lysed with 30 strokes with a 2 ml dounce homogenizer on ice. lysed cells were then subject to centrifugation at 1000xg for 5 min at 4 °c, and the post-nuclear supernatants ( pns ) were transferred to new tubes on ice. the α - flag m2 magnetic beads were washed with 1 ml kpbs buffer with the protease inhibitors and resuspend in the same buffer. resuspended bead slurry was added to each pns and incubated at 4 °c for 1 h with gentle rotation. beads were eluted by addition of 120 µl 0. 5 % np - 40 in kbps with the protease inhibitors and incubated for 30 min at 4 °c with gentle rotation, followed by immunoblot analysis. the interaction between protein and rna was assessed using the magna rip rna - binding protein immunoprecipitation kit ( millipore co., burlington, ma ), according to the manufacturer ’ s instructions. briefly, specific antibod ies attached to magnetic beads were incubated with cell lysates.