title: GSM8975476: WT whole cell rep 1; Homo sapiens; RNA-Seq
library_construction_protocol: RNA isolation for lysosomal enrichment experiments Samples were purified using Trizol LS (Invitrogen 10296010) and the RNA clean and concentrator (Zymo R1015 or R1017). Briefly, 750 uL Trizol LS was mixed with 250 uL of sample (if the sample was less than 250 uL, water was added to 250 uL). Samples were homogenized by pipetting and frozen at -80°C if the RNA purification was not done the same day. After thawing, 200 uL chloroform was added per 750 uL of Trizol LS. Samples were mixed by shaking for 15 s and incubated at room temperature for 3 min. Then, samples were centrifuged for 15 min at 12,000 x g at 4°C. 500 uL of the upper aqueous phase was transferred to a new tube, 500 uL of 100% ethanol was added to the sample, and they were mixed by pipetting. 700 uL of this sample was then transferred to zymo-spin IC or IICR column in a collection tube and centrifuged at 10,000 x g for 30 s. The flow through was discarded. Additional sample was transferred to the same column and centrifuged at 10,000 x g for 30 s. Flow through was again discarded. 400 uL of RNA prep buffer was added to the column and centrifuged at 10,000 x g for 30 s. Flow through was discarded. 700 uL of RNA wash buffer was added to the column and centrifuged at 10,000 x g for 30 s. Flow through was discarded. 400 uL of RNA wash buffer was added to the column and centrifuged at 10,000 x g for 30 s. Flow through was discarded. The column was transferred to a new tube for elution. 15 or 25 uL of water was added to the column matrix and the sample was centrifuged at 10,000 x g for 30 s.  5EU labeling and isolation of labeled RNA 150 mL of cells were incubated with 0.5 mM 5EU for 10 min shaking at 37°C, then spun at 300 x g for 3 min, and resuspended in 50 mL of media with 1 mM uridine (~20 min until 5EU was removed). Cells were then spun 2 more times as above resuspending in 50 mL media with 1 mM uridine, and then 200 mL media with 1 mM uridine.  Then cells were incubated at for 2 h post initial wash, with shaking at 37°C. At this point Torin1 (Selleck Chemicals S2827) treatment was started on half of the samples. Timepoints were collected by spinning down 15 mL cells at 300 x g for 3 min and resuspending in 4 mL of Tri reagent (Thermo Fisher Scientific AM9738) and freezing at -80°C.  Cells in tri-reagent were thawed and had 1 ng GFP and 5 ng of nLuc spike-in RNAs added to them. Spike-in RNAs were made by in vitro transcription as in Eisen et al.21. RNAs were then precipitated by adding 100 uL BCP for each 1 mL of tri reagent, mixing by shaking for 15 s, incubation at room temperature for 5 to 15 min, and then centrifugation at 12,000 x g for 15 min at 4°C. The aqueous phases (490-500 ul) were transferred to a fresh tube and 1 uL of 5 mg/mL linear acrylamide was added to each sample. 500 ul of isopropanol was then added per 1 mL of Tri reagent solution used for the sample. The samples were then mixed by vortexing and incubated at -20°C overnight. Samples were centrifuged at 12,000 x g for 8 min at 4°C, and the supernatant was removed without disturbing the pellet. Samples were washed with 1 mL cold 75% ethanol, then centrifuged at 12,000 x g for 5 min at 4°C. Ethanol was removed, and samples were centrifuged again briefly to remove any residual ethanol. Samples were air dried for 5 min and then dissolved in 100 ul water or 150ul water for the four untreated 28 h timepoints.  Biotinylation reactions were done as Eisen et al. 21. Briefly, half of the isolated RNA from above was incubated in a 50 mM HEPES, pH 7.5, 4 mM disulfide biotin azide (Click Chemistry Tools), 2.5 mM CuSO4, 2.5 mM Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA, Sigma-Aldrich), and 10 mM sodium ascorbate (Sigma-Aldrich) in a final volume of 180 uL for 1 h at room temperature protected from light. Reactions were stopped by adding EDTA to a final concertation of 5 mM, brought to 300 uL with water, and then had 300 uL phenol:chloroform:isoamyl alcohol (25:24:1,Sigma P2069-100ML) added. Samples were mixing by vortexing for 20 s, incubated at room temperature for 10 min, and centrifuged at 21,000 x g for 10 min at 4°C. The upper aqueous phase (280 uL) was transferred to a new tube, and 18 uL of 5 M NaCl, 2 uL 5 mg/mL linear acrylamide, and 900 uL ethanol were added, mixed, and incubated at -20°C overnight. Samples were then centrifuged at 21,000 x g for 10 min at 4°C and the supernatant was removed without disturbing the pellet. Samples were washed with 1 mL cold 75% ethanol, then centrifuged at 21,000 x g for 5 min at 4°C. Ethanol was removed, and samples were centrifuged again briefly to remove any residual ethanol. Samples were air dried for 5 min and then dissolved in 100 ul water.  RNA was fragmented for 7 min at 94°C using the NEB RNA fragmentation module (E6150S) in a final volume of 110 uL, and 10 uL of 3M pH 5.5 sodium acetate, 2 uL 5 mg/mL linear acrylamide, and 325 uL ethanol were added, mixed, and incubated at -20°C overnight. Samples were then centrifuged at 21,000 x g for 15 min at 4°C and supernatant was removed without disturbing the pellet. Samples were washed with 1 mL cold 70% ethanol, then centrifuged at 21,000 x g for 10 min at 4°C. Ethanol was removed, and samples were centrifuged again briefly to remove any residual ethanol. Samples were air dried for 5 min and then resuspended in 100ul 1X HSWB (10 mM Tris pH 7.5, 1 mM EDTA, 100 mM NaCl, 0.01% Tween-20).  Purification of biotinylated RNAs were done as Eisen et al. 21. Dynabeads MyOne Streptavidin C1 beads (Invitrogen 65002) for each set of samples were combined and batch washed, with 200 μL of beads per reaction. Beads were washed twice with 1X B&W buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 M NaCl and 0.005% Tween-20), twice with solution A (0.1 M NaOH, 50 mM NaCl), twice with solution B (0.1 M NaCl), and then twice with water, using for each wash a volume equal to that of the initial bead suspension. Following the last wash, beads were resuspended in an initial bead volume of 1X high salt wash buffer (HSWB, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.1 M NaCl, 0.01% Tween-20) supplemented with 0.5 mg/mL yeast RNA (Thermo Fisher) and incubated at 23°C on a thermal mixer, shaking in a cycle of 15 s 1400 RPM on and 1 min 45 s off, for 30 min, again using a volume equal to that of the initial bead suspension. Beads were then washed three times with 1X HSWB per reaction and split for each reaction during the last wash. After the wash was removed, sample RNA previously resuspended in 100 μL 1X HSWB was added to blocked beads and incubated at 23°C on a thermal mixer, shaking in a cycle of 15 s 1400 RPM on and 1 min 45 s off, for 30 min. Beads were washed twice with 400 μL water at 50°C, incubating at 50°C for 2 min for each wash, and then twice with 400 μL 10X HSWB, incubating at 50°C for 2 min for each wash. RNA was eluted from beads by incubating with 180 μL 0.5 M tris(2-carboxyethyl)phosphine (TCEP, Sigma-Aldrich) at 50°C shaking on a thermal mixer in a cycle of 15 s 1400 RPM on and 1 min 45 s off, for 30 min. The initial eluate was collected, and beads were resuspended in 150 μL water and eluted again, combining the two eluates for each sample. RNA from the eluate was then mixed with 22 uL 5 M NaCl, 3 uL 5 mg/mL linear acrylamide and 1 mL ethanol and incubate at -20°C overnight. Samples were then centrifuged at 21,000 x g for 30 min at 4°C and supernatant was removed without disturbing the pellet. Samples were washed with 1 mL cold 75% ethanol, then centrifuged at 21,000 x g for 10 min at 4°C. Ethanol was removed, and samples were centrifuged again briefly to remove any residual ethanol. Samples were air dried for 5 min and then resuspended in 20 ul water.  Samples were then PNK treated as done above for lysosomal enrichment RNA sequencing, brought up to 350 uL with water, and mixed with 350 uL of phenol:chloroform:isoamyl alcohol (25:24:1,Sigma P2069-100ML). Samples were mixing by vortexing for 20 s, incubated at room temperature for 10 min, and centrifuged at 21,000 x g for 10 min at 4°C. The upper aqueous phase (340 uL) was transferred to a new tube, and 340ul chloroform was mixed in before incubating at room temperature for 10 min and centrifugation at 21,000 x g for 10 min at 4°C. The upper aqueous phase (280 uL) was transferred to a new tube, and 18 uL of 5M NaCl, 2 uL 15 mg/mL glycoblue, and 900 uL ethanol were added, mixed, and incubated at -20°C overnight. Samples were then centrifuged at 21,000 x g for 30 min at 4°C and supernatant was removed without disturbing the pellet. Samples were washed with 1 mL cold 75% ethanol, then centrifuged at 21,000 x g for 10 min at 4°C. Ethanol was removed, and samples were centrifuged again briefly to remove any residual ethanol. Samples were air dried for 5 min and then dissolved in 20ul water. 50 ng of RNA, or 10ul for mock IP samples, were then input into the TGIRT RNA sequencing protocol outlined above. Purified RNA (ranging from 100 to 2000 ng) from whole-cell and lysosomal samples had equal amounts of SIRV-Set 3 (Iso Mix E0/ERCC) (Lexogen 051.01) spike-ins added to them such that spike-ins would represent ~0.01 to ~0.2% of reads. They were then treated with DNAse I (Zymo E1010) according to Zymo's instructions. RNA was then purified using Trizol LS and zymo-RNA clean and concentrators kits as above. Then, RNA was fragmented for 7 min at 94°C using NEB RNA fragmentation module (E6150S), before again purifying RNA using Trizol LS and zymo-RNA clean and concentrators kits as above. Then, to remove any 3′ cyclic phosphates from fragmentation, 30 uL of RNA was PNK treated with 50 U T4 PNK (Biosearch technologies P0503K) in a final volume of 60 uL prior to again purifying RNA using Trizol LS and zymo-RNA clean and concentrators kits as above. RNA was then quantified and checked for proper fragmentation by bioanalyzer as outlined above.  RNA sequencing libraries from these samples were prepared based on Xu et al.38 with some modifications. Briefly, reverse transcription was done using TGIRT-III from the Lambowitz lab using an annealed R2 RNA (rArArGrArUrCrGrGrArArGrArGrCrArCrArCrGrUrCrUrGrArArCrUrCrCrArGrUrCrArC/3SpC3/) / R2R DNA (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTN) mix. DNA was cleaned up using MinElute reaction cleanup kits (Qiagen 28204). Then cDNA was ligated to adenylated (NEB E2610S) R1R DNA (/5Phos/NNNNNNNNNNGATCGTCGGACTGTAGAACTCTGAACGTGTAG/3SpC3/) using Thermostable 5′ AppDNA/RNA ligase (NEB M0319S). DNA was cleaned up using MinElute reaction cleanup kit.  cDNA was amplified using Phusion (Thermo Fisher F531S) and the following primers: P5 barcode primer: AATGATACGGCGACCACCGAGATCTACAC-BARCODE-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTT, P7 barcode primer: CAAGCAGAAGACGGCATACGAGAT-BARCODE- CTACACGTTCAGAGTTCTACAGTCCGACGATC. A cycle course of (6, 9, 12, 15, or 18 cycles) or (8, 10, 12, 14, or 16 cycles) was performed on half of the cDNA to determine the fewest amount of cycles necessary for library amplification (established by the PCR product being visible on an ethidium bromide stained agarose gel). Note, that here the primers used are different than those in the Lambowitz protocol. This is for two reasons. One is to use the non-truncated p5 sequence, and the other is to invert the reads such that the base biased sequence near the barcode is read second. If this is not done, sequencing quality is dramatically diminished, because NovaSeq is highly sensitive to base bias in the first 26 cycles.  PCR products were cleaned up to remove primers using 2 rounds of ampure xp bead (Beckman Coulter A63880) with a 1.4 x ratio of beads to PCR volume prior to submission for paired end 50nt sequencing on NovaSeq. As a result of the differences in primer design outlined above, sequencing was done with custom oligos. Read 1 primer was oGJR992: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTT. Read 2 primer was oGJR993: CTACACGTTCAGAGTTCTACAGTCCGACGATC. i7 sequencing primer was oGJR994: GATCGTCGGACTGTAGAACTCTGAACGTGTAG. i5 sequencing primer was oGJR995: GATCGGAAGAGCACACGTCTGAACTCCAGTCAC.