title: GSM5465908: Sample 79_Mouse_Small-intestine_polyA-RNA_Treated_rep1; Mus musculus; RNA-Seq
library_construction_protocol: [dataset3] Sample 41-88: Total RNA isolation: mouse tissues were weighted and homogenized in TRIzol reagent (Invitrogen) till no visible chunks were left; cellular total RNA was isolated according to TRIzol reagent manufacturer's protocol, followed by isopropanol precipitation. PolyA+ RNA isolation: Dynabeads™ mRNA DIRECT™ Purification Kit (Invitrogen™) was used for polyA+ RNA enrichment, from the purified total RNA. [dataset4] Sample 89-136: Chromosome-associated RNA (caRNA) isolation: cell pellet from mouse tissues were lysed with ice cold 1X lysis buffer (10 mM Tris-HCl, pH = 7.5, 0.05% NP40, 150 mM NaCl), and then incubated on ice for 5 min. Gently pipet the cell lysate over 2.5 volumes of chilled sucrose cushion (24% RNase-free sucrose in lysis buffer) and spin at 4°C with 15,000 g for 10 min. The supernatant was carefully collected as cytoplasmic fraction. Gently rinse the nuclei pellet with ice cold PBS/1mM EDTA without dislodging the pellet, and then aspirate the PBS/EDTA. Nuclei pellet were resuspended in an ice cold glycerol buffer (20 mM Tris-HCl, pH = 7.9, 75 mM NaCl, 0.5 mM EDTA, 0.85 mM DTT, 0.125 mM PMSF, 50% glycerol), followed by adding a proper volume of nuclei lysis buffer (10 mM HEPES, pH = 7.6, 1 mM DTT, 7.5 mM MgCl2, 0.2 mM EDTA, 0.3 M NaCl, 1.5 M UREA, 1% NP-40) and vortex for 2 rounds (5 seconds per round). The nuclei pellet suspension was incubated for 2 min on ice, and then centrifuged at 4 °C with 15,000 g for 2 min. The soluble nuclear fraction/nucleoplasm was collected as the supernatant, while the chromosome-associated fraction was harvested as the pellet which was further rinsed by ice cold PBS/1mM EDTA. To minimize rRNA components in caRNA fraction, two rounds of RiboMinus™ Eukaryote System v2 (Invitrogen™) was further used for rRNA removal. [dataset3] Sample 41-88: Mouse tissues, except immune cells, were collected from two replicates (one wild-type mice per replicate) with age and sex matched; to harvest enough materials, mouse immune cells such as B cells, CD4 T cells and CD8 T cells were collected and combined from two replicates (8 wild-type mice per replicate) with age and sex matched. After extracting the polyA+ RNA from mouse tissues, ~50-200 ng RNA was fragmented into 50-80 nt length using Na2CO3/NaHCO3 buffer (pH 9.2) and heated at 95 °C for 3 min, followed by purification with Oligo Clean & Concentrator (Zymo Research). 3'- and 5'-end repairing was conducted with T4 Polynucleotide Kinase (EK0032, Thermo Fisher Scientific). RNA was mixed with 5 µL of 10X T4 Polynucleotide Kinase Reaction Buffer (B0201S, NEB) and 5 µL of T4 PNK, diluted to a final volume of 50 µL, and incubated at 37 °C for 1 hour; then 2 µL of T4 PNK and 5 µL 10 mM ATP were added for another incubation 37 °C for 30 min, followed by Oligo Clean & Concentrator purification. To perform 3'-adaptor ligation, ~6 µL repaired RNA fragments were incubated with 1.0 µL 3'-Adaptor (5'App-NNNNN ATCACG AGATCGGAAGAGCACACGTCT-3SpC3, with ATCACG as the inline barcode) at 70 °C for 2 mins and immediately placed on ice. 10 µL 3´ Ligation Reaction Buffer (2X) (NEB) and 3 µL 3´ Ligation Enzyme Mix (NEB) were added to the RNA-adapter mixture. The reaction was incubated at 25 °C for 2 hours followed by 16 °C for 10 hrs. Then 1.0 µL SR RT Primer for Illumina (NEB) was added to the mixture, followed by 5 minutes at 75°C, 15 minutes at 37°C, 15 minutes at 25°C, and then on ice. The denatured 1.0 µL 5'-Adaptor (5'-GUUCAGAGUUCUACAGUCCGACGAUC NNNNN-3') was added into the mixture, together with 1.0 µL 5´ Ligation Reaction Buffer (10X) (NEB) and 2.5 µL 5´ Ligation Enzyme Mix (NEB). The reaction was incubated at 25 °C for 4 hours, followed by RNA Clean & Concentrator purification  ~10 ng of the purified RNA was saved as “input” and the remainder was subjected to BID-seq optimized bisulfite treatment, as the “treated”. Then both the “input” and “treated” samples were mixed with 4 µL 5X SSIV Buffer (Thermo Scientific), 2 µL of 10 mM dNTP Solution Mix (NEB), 1 µL 100 mM DTT, 1 µL of RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Scientific), and 1 µL of SuperScript IV Reverse Transcriptase (Thermo Scientific). The reaction was diluted to 20 µL and incubated at 50 °C for 30 mins. 1.0 µL of RNase H (NEB, M0297L) was then added and the reaction was incubated at 37 °C for 20 mins. The cDNAs were purified by Oligo Clean & Concentrator (Zymo Research). The libraries were amplified using the SR Primer for Illumina (NEB) and indexed primers (from NEBNext Multiplex Oligos for Illumina). [dataset4] Sample 89-136: Mouse tissues, except immune cells, were collected from two replicates (one wild-type mice per replicate) with age and sex matched; to harvest enough materials, mouse immune cells such as B cells, CD4 T cells and CD8 T cells were collected and combined from two replicates (8 wild-type mice per replicate) with age and sex matched. After extracting the caRNA from mouse tissues, ~50-200 ng RNA was fragmented into 50-80 nt length using Na2CO3/NaHCO3 buffer (pH 9.2) and heated at 95 °C for 3 min, followed by purification with Oligo Clean & Concentrator (Zymo Research). 3'- and 5'-end repairing was conducted with T4 Polynucleotide Kinase (EK0032, Thermo Fisher Scientific). RNA was mixed with 5 µL of 10X T4 Polynucleotide Kinase Reaction Buffer (B0201S, NEB) and 5 µL of T4 PNK, diluted to a final volume of 50 µL, and incubated at 37 °C for 1 hour; then 2 µL of T4 PNK and 5 µL 10 mM ATP were added for another incubation 37 °C for 30 min, followed by Oligo Clean & Concentrator purification. To perform 3'-adaptor ligation, ~6 µL repaired RNA fragments were incubated with 1.0 µL 3'-Adaptor (5'App-NNNNN ATCACG AGATCGGAAGAGCACACGTCT-3SpC3, with ATCACG as the inline barcode) at 70 °C for 2 mins and immediately placed on ice. 10 µL 3´ Ligation Reaction Buffer (2X) (NEB) and 3 µL 3´ Ligation Enzyme Mix (NEB) were added to the RNA-adapter mixture. The reaction was incubated at 25 °C for 2 hours followed by 16 °C for 10 hrs. Then 1.0 µL SR RT Primer for Illumina (NEB) was added to the mixture, followed by 5 minutes at 75°C, 15 minutes at 37°C, 15 minutes at 25°C, and then on ice. The denatured 1.0 µL 5'-Adaptor (5'-GUUCAGAGUUCUACAGUCCGACGAUC NNNNN-3') was added into the mixture, together with 1.0 µL 5´ Ligation Reaction Buffer (10X) (NEB) and 2.5 µL 5´ Ligation Enzyme Mix (NEB). The reaction was incubated at 25 °C for 4 hours, followed by RNA Clean & Concentrator purification  ~10 ng of the purified RNA was saved as “input” and the remainder was subjected to BID-seq optimized bisulfite treatment, as the “treated”. Then both the “input” and “treated” samples were mixed with 4 µL 5X SSIV Buffer (Thermo Scientific), 2 µL of 10 mM dNTP Solution Mix (NEB), 1 µL 100 mM DTT, 1 µL of RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Scientific), and 1 µL of SuperScript IV Reverse Transcriptase (Thermo Scientific). The reaction was diluted to 20 µL and incubated at 50 °C for 30 mins. 1.0 µL of RNase H (NEB, M0297L) was then added and the reaction was incubated at 37 °C for 20 mins. The cDNAs were purified by Oligo Clean & Concentrator (Zymo Research). The libraries were amplified using the SR Primer for Illumina (NEB) and indexed primers (from NEBNext Multiplex Oligos for Illumina). All libraries were sequenced on Illumina NovaSeq 6000 with single-end 100 bp read length.