Supplementary Figure 1. Brwd1 transcripts in Brwd1 mut B cells. Nature Immunology: doi: /ni.3249

Transcription

1 Supplementary Figure 1 Brwd1 transcripts in Brwd1 mut B cells. (a) Analysis of Brwd1 transcripts in isolated small pre-b (Lin B220 + CD43 IgM FSC lo ) and splenic B (B220 + ) cells from WT and Brwd1 mut bone marrow (BM) and spleen by PCR using cdnas prepared from those cells with several Brwd1 primer sets (Supplementary Table 1) that amplify different regions of Brwd1 transcript. (b) DNA sequence chromagrams from homozygous WT and Brwd1 mut animals across the exon 10 intron 10 junction, as indicated. There is a T--C change in the Brwd1 mut allele (n = 10). (c) Sequencing of aberrant PCR products obtained with primer set 2 (Supplementary Table 1) that amplifies exon 9 11 of cdna prepared from isolated small pre- B cells of WT and Brwd1 mut BM with predicted translated amino acid sequences (n = 5). (d) Immunoblot analysis of BRWD1 on tal nuclear cell lysates from splenic B cells isolated from WT and Brwd1 mut mice (n = 3).

2 Supplementary Figure 2 Early common progenir, myeloid, erythroid and T lymphocyte cell development is unaltered in Brwd1 mut (Mut) animals. (a) Absolute numbers of cells per mouse at different stages of B cell development in the BM of WT and Brwd1 mut/wt heterozygous (Het) mice (n = 3). (b) Differential blood counts of WT and Brwd1 mut mice (n = 3). (c) Flow cymetry analysis of common myeloid progenirs (CMP; Lin Sca1 ckit hi Fc R lo CD34 hi ), granulocyte and macrophage progenirs (GMP; Lin Sca1 ckit hi Fc R hi CD34 hi ) and megakaryocyte and erythroid progenirs (MEP; Lin Sca1 ckit hi Fc R lo CD34 lo ) from BM of WT and Brwd1 mut mice (n = 3). (d) Cellularity of myeloid progenir cells in the BM of WT and Brwd1 mut mice (n = 3). (e) Flow cymetric analysis of BM erythroid cells of WT and Brwd1 mut mice (n = 3). (f) Cellularity of erythroid cells at different developmental stages includes proerythroblasts (population I, Ter119 med Cd71 hi ), basophilic erythroblasts (population II, Ter119 hi Cd71 hi ), and late erythroblasts (population III IV, Ter119 hi Cd71 med, Ter119 hi Cd71 lo ) (n = 3). (g) Flow cymetric analysis of BM myeloid cells of WT and Brwd1 mut mice (n = 3). (h) Cellularity of Gr1 + Mac1 + cells in the BM of WT and Brwd1 mut mice (n = 3). (i) Flow cymetric analysis identifying LSK (Lineage Sca1 + ckit + ), HSC (hemapoietic stem cells), MPP (multipotent progenirs), LMPP (lymphoid restricted multipotent progenirs) and CLP (common lymphoid progenirs) in the BM of WT and Brwd1 mut mice (n = 3). (j) Total progenir cell numbers of LSK, HSC, MPP, LMPP and CLP progenirs in BM of WT and Brwd1 mut mice (n = 3). (k) Flow cymetric analysis of thymocytes identifying SP (single-positive, CD4 + or CD8 + ), DP (double-positive, CD4 + CD8 + ) and DN (double-negative, CD4 CD8 ) populations in WT and Brwd1 mut mice. The DN population was further analyzed by CD25 and CD44 staining identify DN1, DN2, DN3 and DN4 populations (right panel of j) (n = 4). (l) Absolute numbers of DNs, CD4 and CD8 populations in the thymus of WT and Brwd1 mut mice (n = 4). All data in bar graphs are presented as average s.d.

3 Supplementary Figure 3 BRWD1 in B cell development and immunoglobulin light chain recombination. (a,b) LSK progenirs from WT (CD45.1) and WT (CD45.2) (a) or WT (CD45.1) and Brwd1 mut (CD45.2) (b) mice were flow sorted and mixed 1:1 and transplanted in sublethally irradiated (550 rad) Rag2 / Il2rg / hosts. B cell development in spleen (a) and T cell development in thymi (b) were then analyzed by flow cymetry after 5 weeks of transplantation. Data are representative of four independent experiments. (c) Flow cymetric assay for apopsis with annexin V and 7-AAD in B cell progenirs of different developmental stages isolated from WT and Brwd1 mut mice (n = 2). (d) Cell cycle analysis of B cell progenirs of different developmental stages isolated from WT and Brwd1 mut mice. Numbers above bracketed lines indicate the percentage of cells in S-G2M (n = 2). (e) Quantitative RT-PCR analysis of the expression of Ccnd2 and Ccnd3 in flow-sorted large pre-b cells from BM of WT and Brwd1 mut mice (n = 2). *P < 0.05 compared with respective controls (unpaired t-test). (f). Frequency (as a percentage) of J usage in rearranged Igk in isolated small pre-b cells of WT and Brwd1 mut mice (n = 2). (g) Usage of V gene family in small pre-b cells of WT and Brwd1 mut mice (n = 2). (h) Analysis of Igl-expressing immature B cells and splenic IgM-expressing B cells isolated from WT and Brwd1 mut mouse BM and spleen, respectively (n = 3).

4 Supplementary Figure 4 H3K9Ac and H3S10pK14Ac hisne marks do not require BRWD1. (a) ChIP with IgG or BRWD1-specific antibodies in flow-sorted small pre-b cells isolated from WT and Brwd1 mut (Mut) mice followed by quantitative PCR for J 1 J 2 and J 4 J 5 regions. Background IgG subtracted from each lane. (b,c) ChIP with IgG, H3K9Ac (b) and H3S10pK14Ac (c) specific antibodies in flow-sorted pro-b and small pre-b cells isolated from WT and Brwd1 mut mice followed by quantitative PCR with non-overlapping primer sets (Supplementary Table 1) designed detect various regions of Igk. Data are representative of three independent experiments (average s.d.).

5 Supplementary Figure 5 De novo motifs in BRWD1, H3K9Ac and H3S10pK14Ac ChIP-Seq peaks. (a) Representative BRWD1, H3K9Ac and H3S10pK14Ac ChIP-Seq alignment (chromosome 1 and chromosome 6). A region (40,000K 110,000K) is zoomed in on (middle panel). Data are representative of two independent experiments. (b) Percentage of ChIP-Seq peaks (P < 10 7 ) alone or in combination in different genomic regions. Promoter ( 5 kb +500 bp from TSS); Intragenic (in exons or introns); (not in promoter, exons and introns). (c) ChIP-qPCR with IgG and BRWD1-specific antibodies in flow-sorted immature B cells from Igk del animals and for indicated regions of Igl (expressing immunoglobulin -chain). Data are representative of two independent experiments (average s.d.). (d) De novo DNA sequence motifs identified among BRWD1, H3K9Ac and H3S10pk14Ac-bound regions. (e,f) De novo DNA sequence motifs identified in BRWD1/H3S10pK14Ac (d) and BRWD1/H3K9Ac/H3S10pK14Ac (e) binding regions.

6 Supplementary Figure 6 Regulation of gene accessibility during B lymphopoiesis. (a) Total and overlapping accessibility peaks in WT and Brwd1 mut small pre-b cells. (b) Accessibility (open chromatin) at Ccnd3 (cyclin D3) locus in WT and Brwd1 mut (Mut) small pre-b cells. Y-axis represents tags per million reads. Data are representative of two independent experiments. (c) Accessibility (open chromatin) at entire Igk locus (mm9 chromosome 6: 67,500,000 70,800,00) showing distal and proximal V regions. Data are representative of two independent experiments. (d) Accessibility (open chromatin) at proximal V and J -C regions. Data are representative of two independent experiments. (e) Accessibility (open chromatin) at J -E i regions and immediate upstream. Data are representative of two independent experiments.

7 Supplementary Figure 7 BRWD1 regulates accessibility and nucleosome positioning. (a) Nucleosome positioning at RSS and J s in WT and Brwd1mut (Mut) small pre-b cells. Data are representative of two independent experiments. (b) Accessibility (open chromatin) and nucleosome positioning at 3ʹE. Data are representative of two independent experiments. (c) DNA footprinting analysis of 1 kb region for WT (red) and Brwd1 mut (blue) small pre-b cells at H3K9Ac, BRWD1+H3K9Ac, H3S10pK14Ac and BRWD1+H3S10pK14Ac peaks centered at 0. Nucleosome differential (upper panel) and accessibility (lower panel) were demonstrated. For nucleosome differential (Y-axis), values greater than 0 indicate the presence of a nucleosome, whereas values less than 0 tend be nucleosome free. The X-axis is the distance in bp from the indicated peak or motif. (d) Alignment of BRWD1, H3K9Ac and H3S10pK14Ac enrichment at GAGA motifs. The Y-axis represents the normalized immunoprecipitation signal distribution for GAGA motifs centered at 0. (e) Position of short GAGAG (CTCTC) motifs in J and E i regions. (f) Quantitative RT-PCR analysis of the expression of Rag1 and Rag2 in flow-sorted small pre-b cells from BM of WT and Brwd1 mut mice (n = 2). (g) Quantitative RT-PCR analysis of the expression of Tcfe2a (encodes E2A), Pax5, Ikzf1 (IKAROS), Irf4, Irf8, Smarca4 and Myc in flow-sorted small pre-b cells from BM of WT and Brwd1 mut mice (n = 3).

8 Supplementary Table 1. List of probes and primers. Quantitative PCR Ccnd3-Fw 5 -CGC TGC GAG GAG GAT GTC TT-3 Ccnd3-Rev 5 -CAA CTG CCA TGG AGC CAC AG-3 Ccnd2-Fw 5 -AGC TGT CCC TGA TCC GCA AG-3 Ccnd2-Rev 5 -GCA GCT CTG TCA GGG CAT CA-3 Brwd1-Fw1 5 -GCCTGGTGTTCAGATGCTGTG-3 Brwd1-Rev1 5 -GCTGTTCCATCTCGGCTACCA-3 Brwd1-Fw2 5 -TTGCTTCTGGCAGTGGGATTT-3 Brwd1-Rev2 5 -GCTTTCAAGCTCGGCGATTT-3 Brwd1-Fw3 5 -GTGGACGGAAACCCTCATCC-3 Brwd1-Rev3 5 -CCATTTGCAGTCCCATCCTG-3 Brwd1-Fw4 5 -GATCCAGCGACTGGCAGACT-3 Brwd1-Rev4 5 -TGGGGTTGATACGGTTCTTGA-3 Brwd1-Fw5 5 - CGCCAATGGATTTTGGAACA-3 Brwd1-Rev5 5 - CACCAGGGACAGCACCGTTA-3 Rag1-Fw 5 -CTG CAG ACA TTC TAG CAC TC-3 Rag1-Rev 5 -AAC TGA AGC TCA GGG TAG AC-3 Rag2-Fw 5 - TCA TAA GTG AGA AGC CTG GT-3 Rag2-Rev 5 -CCT TCA GTG CCA AAA TAA GA-3 Tcfe2a-Fw 5'-TCCTTTGACCCTAGCCGGACATAC-3' Tcfe2a-Rev 5'-CCAACACTGGTGTCTCTCCCAAAG-3' Pax5-Fw 5'-CGCGTGTTTGAGAGACAGCACTACT-3' Pax5-Rev 5'-GTCTCGGCCTGTGACAATAGGGTAG-3' Ikzf1-Fw 5'-CACTACCTCTGGAGCACAGC-3' Ikzf1-Rev 5'-ATAGGGCATGTCTGACAGGCA-3' Irf4-Fw 5'-CTACCCCATGACAGCACCTT-3' Irf4-Rev 5'-CCAAACGTCACAGGACATTG-3' Irf8-Fw 5'-GAGCGAAGTTCCTGAGATGG-3' Irf8-Rev 5'-TGGGCTCCTCTTGGTCATAC-3' Smarca4-Fw 5'-AGAAGCTGATTCCTCCGCAAC-3' Smarca4-Rev 5'-AAGCCTGTACTCCCGCTCTTG-3 Myc-Fw 5 -GCCCCCAAGGTAGTGATCCT-3 Myc-Rev 5 -GTGCTCGTCTGCTTGAATGG-3 B-2 Microglobulin-F 5 -AGACTGATACATACGCCTGCA-3 B-2 Microglobulin-R 5 -GCAGGTTCAAATGAATCTTCA-3 Igk-Germline-F 5 -GAGGGGGTTAAGCTTTCGCCTACCCAC-3 Igk-Germline-R 5 -GTTATGTCGTTCATACTCGTCCTTGGTCAA-3 ChIP- Quantitative PCR Vκ F 5 - TTGTGCTCACCCAATCTCCAG-3 Vκ R 5 - GGCTGTCCTGGTTTCTGTTGG-3 Jκ 1- Jκ 2 -F 5 -AGGCACCAAGCTGGAAATCAA-3 Jκ 1- Jκ 2 -R 5 -TGCCTTGGAGAGTGCCAGAAT-3 Jκ 4- Jκ 5 - F 5 - GCTCGGGGACAAAGTTGGAA-3

9 Jκ 4- Jκ 5 -R 5 - GATGCACAGGTTGCCAGGAAT-3 Jκ 5 - Eκi- F 5 - TGTTTTAATGGCCACGGTTTTG-3 Jκ 5 - Eκi- R 5 - AGAAGAGGTTGCGGACCGTTT-3 Eκi- F 5 - GCAGGTGGCCCAGATTACAGT-3 Eκi- R 5 - AGGGCCTTAAGCCAGGGTCT-3 Cκ-F 5 - TCTGGAGGTGCCTCAGTCGT-3 Cκ- R 5 -CCAACTGTTCAGGACGCCATT-3 3 Eκ- F 5 - CCCCACCTCCATCTTGTTTGA-3 3 Eκ- R 5 - GGGCCCAGTGACCATATCAGA-3 Actin γ1-f 5 -CCTCCTCCAATAAAGGGACA-3 Actin γ1-r 5 -GCCATCACATCCCAGTCA-3 β-globin-f 5 -GCTGCTGGTTGTCTACCCTT-3 β-globin-r 5 -GCAGAGGCAGAGGATAGGTC-3 Eλ1-3-F 5 -AAGCTCTGTGGAGGAGGTTG -3 Eλ1-3-R 5 -AGCTTGTGGACTCTCAAGGG-3 Eλ2-4-F 5 -TAGGTTGGGGCAGAGAGATG-3 Eλ2-4-R 5 -GCTTGTGGATTCTCAAGGGT-3 IgλJ1-F 5 -GATCTTTCAGTGATGTCACCACC-3 IgλJ1-R 5 -GCACCTCAAGTCTTGGAGAGAAC-3 IgλJ2-F 5 -CCACCCACTGCTTCTCAAGTG-3 IgλJ2-R 5 -AGACAACAAGGGCTGGGCTTA-3 Short Hairpin RNA Constructs shrna-brwd1-5 -CGGATCTGTATCACTAGAGAA-3 4 (target seq) shrna-brwd1-5 -GCTGTTGACAGTGAGCGCCGGATCTGTATCACTAGAGAATAGTGAAGCC 4 (97mer) ACAGATGTATTCTCTAGTGATACAGATCCGTTGCCTACTGCCTCGGA -3' Kappa Recombination degvκ 5 -GGC TGC AGS TTC AGT GGC AGT GGR TCW GGR AC-3 MAR (Igκ intron) 5 -AAC ACT GGA TAA AGC AGT TTA TGC CCT TTC-3 κ-meth-f 5 -ATG ACC CAG AGG ATG AAA C-3 κ-j1-r 5 -AGC ATG GTC TGA GCA CCG AGT AAA GG-3 Eκi-F 5 -AGA AGT GAA GTC TGC CAG TT-3 Eκi-R 5 -GTA ACC ACA TGG GAC AAT TT-3 Jκ Usage Vκ-FW Cκ 5 -AGCTTCAGTGGCAGTGGRTCWGGRAC-3 5 -CTTCCACTTGACATTGATGTC-3

10 Supplementary Table 2. ATAC-seq alignment statistics. WT1 WT2 Mut1 Mut2 Raw reads-pair Raw reads - Pair Trimmed reads - Pair % % % % Trimmed reads - Pair % % % % Complete pairs after trimming % % % % Aligned reads % % % % High quality, unique aligned reads % % % % High quality, unique, paired alignments % % % % From WT and Brwd1 mut (Mut) small pre-b cells, duplicate samples were obtained. The WT samples had x 10 8 aligned reads (~97.5% of tal sequences) and Brwd1 mut samples had x 10 8 aligned reads (~97.7%). ATAC-Seq profiles were highly reproducible between biological replicates (Spearman s r=0.99).

11 Supplementary Table 3. Cryptic RSS (crss) and GAGA motifs in open+6-chip-seq peaks (P<10-7 ). Sequence under common region of peaks Location Gene Where the peak is AGGAATGTCGAAATTACTGAAAAACGTGAAAAATGAGAAATGCACACTGCAG chr4 Cdk5rap2 Intron 4 of GACCTGGAATATGGCGAGAAAACTGAACATCACGGAAAATGAGAATAACACT (Protein 36 CTTTAGGTAGTGAAATATGACGAGAAATATTGAAAAATAAGC To coding) Chromatin remodeling (Nucleosome shifting) CCAACGGATGTGTTTTTCAGTGTAACTCACTCATCTAATATGTTCTACAGTGT GGTTTTTTATCATTTTCCATGTTCCTCATTGTAACTCATTGATATACACTGTTC TACAATTCCCGTTTCCAACGAATGTGTTTTTCAGTGTAACTCACTCATCTAATA TGTTCTACAGTGTGGTTTTTATCATTTTCCATGTTTCTCATTGTAACTCATTGA TATACACTGTTCTACAAATCCCGTTTCTATA (RIC score )- RSS12 sequence; + strand chr Chl (Protein coding) Intron 4 of 26 No NNNNGATCCTACAGTGTGCATTTCTCATTTTTCACGTTTTTCAGTGATTTCGT CATTTTTCAAGTCATCAAGTGGCTGTTTCTCATTTTCCATGATTTTCAGTTTTC TTGCCATATTCCTTGTCCTACAGTGGACATTTCTAAATTTTCCACCTTTTTCAG TTTTCCTTGTCATATTTCAGGTCCTACAGTGTGTATTTCTCATTTTTCACGTTT TTCATTGATTTCGTCATTTTTCAAGTTGTCAAGTGCATGTTTCTCATTTTCCAT GATTTTCAGTTTTCTTGCCATA chr To Mir101c miscrna No GTGTATTTCTCATTTTCCGTGATTTTCAGTTTTCTCGACATATTCCAGGTCCTA CAGTGTGCATTTCTCATTTTTCACGTTTTTCAGTGATTTCGTCATTTTTCACGT CGTCAAGTGGATGTTTCTCATTTTCCATGATTTTCAGTTTTCTTGCCAT chr Mir101c No TATTTCACGTCCTAAAGTGTGTATTTCTCATTTTCCGTGATTTTCAGTTTTATC GCCAGATTCCAGGTCCTACGGTGTGCA chr Mir101c No

12 TGGAAAATGATAAAAACCACACTGTAGAACAGAGTAGATGAGTGAGTTACAC TGAAAAACACAATTCGTTGGAAACGGGAATTGTGTATATCAATGAGTTACTAG GAGAAACATGGAAAATGATAAAAAC chr Dpy19l1 (Protein coding) No TTTTGAGGTGCACACTGAAGGACCTGGAATTATGCGAGAAAACTGAAAATCA CGGAAAATGAGAAATACACACTTTAGGACGTGAAAAATGGCGAGGAAAACTG AAAAAGGTGGAAAATTTAGAAATGTCCTCTGTAGGACATGGAATATGGCAAG AAAACTGAAAATCATGGAAAATGAGAAACATCCACTTGATGACTTGAAAAATG ACGAAATCATTAAAAAACGTGAAAAATGAGAAATGCCCACTGAAGGACCTGG AATATGGGGAGAAAACTGAAAATCACGGAAAATGAGAAATACACACTTTAGG ACGTGAAATATGGCGAGGAAAACTGAAAAAGGTGGAATATTTAGAAATGTCC ACTGTAGGACGTGGAATATAAGT chr Rik A05 (Nucleosome Depletion) GTTTTACCTACATTGTTCCAACATGCCAGAGGCTGTTCACCTTGGAGACCTG CTGCGGATATGGGTACGGCCCGGAGATTTACAAGCTCTCCCCCGGATTTTCA AGGGCCAGCGAGAGCTCACCGGATGCCGCCGGAACCATGACGCTTTCCAA GGCACGTGCCCCTCTCTCAGGGCGAACCCATTCCAGGGCGCCCTGCCCTTC ACAAAGAAAAGAGAACTCTCCCCGGGGCTTCTCCGGGATCGGTCGCGTTAC CGCACTGGACGCCTCGCGGCGCCCATCTCCGCCACTCTGGATTTGGGGATC TGAACCCGACTCCCCTTCAATCGGCCGAGGGCAACGGAGGCCATCGCCCAT CCCTTCGGAACGGCACTCACCCATCTCTCAGGACTGACTGACCCATGTTTAA C chr Axin2 (Protein coding), Nucleosome shifting and depletion CTGAAAATCATGGAAAATGAGAAACATCCACTTGACGACTTGAAAAATGACAA AATCACTGAAAAAGGTGAAAAATGAGAAATGCACACTGTAGGACTTGGAATA TGGCGAGAAAACTGAAAATCACGGAAGTGAGGG chr Rik B15 Nucleosome depletion AGAGTCTGCTATTTCAAAGCATAAGGAAAAAGTAGGAGAAAAACGTGAGGCT GTTTGTGGATGGTCGAGGCTGCTTTAGGGAGCCTCCTCACCATTCTGCACTT GCAAACCGGGCCACTAGAACCCGGTGAAGGGAGAAACCAAAGCGACCTGAA ACAATAGGTCACATGAAGGCCAGCCACCTCCATCTTGTTGTGCAGGAGTTCA GTTAGCAGACAAGATGGCTGCCATCCACATGTCACCTTTCATCTTGGTGAGG TCAATGTGCAGCCGAGTGACAGGACAAGGAAGTAGACATGCAGACAACAGA CATGCAGGCGAACCACCTCCCTTCTGTGTTTGGATAAAAGACATACAACAAT TTTTATTTTTTACAGTAAGCCTTAAAAAGCACTCTGACAGCCACTCAGATATCT ACCTTCTATGT chr To Ly6c2 (Protein coding) Nucleosome depletion

13 CTCTAGATAACCTCGGGCCGATCGCACGCCCCCGTGGCGGCGACGACCCAT TCGAACGTCTGCCCTATCAACTTTCGATGGTAGTCGCCGTGCCTACCATGGT GACCACGGGTGACGGGGAATCAGGGTTCGATTCCGGAGAGGGAGCCTGAG AAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCACTCC CGACCCGGGGAGGTAGTGACGAAAAATAACAATACAGGACTCTTTCGAGGC CCTGTAATTGGAATGAGTCCACTTTAAATCCTTTAACGAGGAT chr To _ + Rn45s Non coding RNA Nucleosome depletion CATAAACGATGCCGACTGGCAATGCGGCGGCGTTATTCCCATGACCCGCCG GGCAGCTTCCGGGAAACCAAAGTCTTTGGGTTCCGGGGGGAGTATGGTTGC AAAGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAGCC TGCGGCTTAATTTGACTCAACACGGGAAACCTCACCCGGCCCGGACACGGA CAGGATTGACAGATTGATAGCTCTTTCTCGATTCCGTGGGTGGTGGTGCATG GCCGTTCTTAGTTGGTGGAGCGATTTGTCTGGTTAATTCCGATAACGAACGA GACTCTGGCATGCTAACTAGTTACGCGACCCCCGAGCGGTCGGCGTCCCCC AACTTCTTAGAGGGACAAGTGGCGTTCAGCCACCCGAGATTGAGCAATAACA GGTCTGTGATGCCCTTAGATGTCCGGGGCTGCACGCGCGCTACACTGACTG GCTCAGCGTGTGCCTACCCTACGCCGGCAGGCGCGGGTAACCCGTTGAAC CCCATTCGTGATGGGGATCGGGGATTGCAATTATTCCCCATGAACGAGGAAT TCCCAGTAAGTGCGGGCCATAAGCTTGCGTTGATTAAGTCCCTGCCCTTTGT ACACACCGCCCGTCGCTACTACCGATTGGATGGTTTAGTGAGGCCCACGGC CCTGGTGGAGCGCTGAGAAGACGGTCGAACTTGACTATCTAGAGGAAGTAA AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAACGGGA GACTGTGGAGGAGCGGCGGCGTGGCTCGCTCTCCCCGTCTTGTGTGTGTCC TCGCCGGGAGGCGCGTGCGTCCCGGG (RIC score )- RSS23 sequence; - strand chr To Rn45s Non coding RNA Nucleosome depletion ATTCACTCACTCATTCCGTCTCTCCCAAGGGTCCTCCCTAGCTCAGGGCGGG GCACTCACGCACACATGACAGAGCAGCGTACCCACCCACCACTTCTCCCGA GTGGCACAAACGGTCCCCATCGTCCCGCCCGAACATAGCGAACACGGGCTG GACTCGGGCACCCAAAAACGAAAGTATGACAGGGAGCTGTGAAAACAGGCG AAATCATT chr To Gm7102 (Predicted protein coding) Nucleosome depletion ACACTGTAGAACATAGTGTGAAATGCACATTGTAAAACACAGTATGTTAATGA GTTGCACTGAAAAACAGAAAATGGGAAATGCACAGTGTAGAACCCAATATAT GAGTGAGTTGCACTGAAACACCTAGAAAATCAGACCGGCACATTGTAGAACA TAGTGTGAAATGCACATTGTAAAACACAGGATGTTAATGAGTTGCACTGAAAA ACATAGAAAATGGGAAATGCACAGTGTAGAACATTGTATATGAGTGCTTTGCA CTGAACAACCTAGAAAAAAAAGA (RIC score ), RSS12 sequence; + strand (RIC score ), RSS23 sequence; + strand chrx Rik A15 Intron 1 of 1 Nucleosome depletion

14 GAGTTTCTCATTGTAACTCATTGATATACACTGTTCTACAATGCCCGTTTCCA ACGTATGTGTTTTTCAGTGTAACTCACTCATCTAATATGTTCTACAGTGTGGTT A (RIC score ), RSS12 sequence; + strand chrx Pak3 (Protein coding) The RIC (RSS Information Content) score goes from (very bad) 0 (very good). It has been shown that there is a good correlation between this score and the RSS functionality. For the current version, pass/fail RIC thresholds are from Cowell et al (2002) as follows: 12 RSS: pass with RIC > ; 23 RSS: pass with RIC > GAGA motifs: (C/G)A(C/G)A(C/G)A. or( G/C)T(G/C)T(G/C)T.) No