Single cell full length RNA sequencing

Abstract

The invention relates to methods for processing an RNA sample and allows for single cell sequencing of full length total RNA. The method includes labeling the RNA sample with at least one of a barcode and a unique molecular identifier.

Claims

1. A method for preparing an sequencing library, preferably a deep-sequencing library, wherein the method comprises the steps of: step a) providing a sample containing RNA; step b) optionally fragmenting the RNA; step c) polyadenylating the, optionally fragmented, RNA; step d) hybridizing a poly-T primer to the polyadenylated RNA and performing reverse transcription of the hybridized RNA thereby obtaining cDNA; and step e) optionally, performing a second strand synthesis, wherein the poly-T primer comprises at least one of an identifier sequence (barcode) and a unique molecular identifier (UMI).

2. The method according to claim 1, wherein the RNA sample is a cellular RNA sample, preferably wherein the RNA sample is a single cell, more preferably wherein the RNA is from a cell nucleus, most preferably from a single cell nucleus.

3. The method according to claim 1, wherein the RNA is a small non-coding RNA, preferably selected from the group consisting of at least one of a microRNA, a snRNA and a snoRNA.

4. The method according to claim 1, wherein the method comprises fragmentation step b), and wherein the fragmentation is step is performed by exposure to a divalent metal-cation at a temperature between about 55-100° C.

5. The method according to claim 4, wherein the divalent metal cation is selected from the group consisting of Mg2+, Mn2+, Ca 2+ and Zn 2+, preferably the divalent metal cation is Mg2+.

6. The method according to claim 1, wherein the method comprises fragmentation step b), and wherein the fragmentation step is followed by an end-repair step to add an OH group at the 3′end of the fragmented RNA prior to polyadenylation step c).

7. The method according to claim 1, wherein the method further comprises at least one of the following steps: step f) in vitro transcription of the cDNA obtained in step d) thereby obtaining amplified RNA (aRNA); and step g) ribosomal-RNA (rRNA) depletion.

8. The method according to claim 1, wherein the method further comprises one or more of the following steps: step h) ligating an oligonucleotide adapter to the aRNA obtained in step f); step i) performing reverse transcription of the, optionally adapter-ligated, aRNA to obtain cDNA; step j) degrading the remaining aRNA; step k) optionally amplifying the cDNA to generate a cDNA library comprising double-stranded cDNA with sequencing primer binding sites; step l) selecting by size the cDNA library obtained in step k); and step m) sequencing the, optionally size selected, cDNA library from step k).

9. The method according to claim 8, wherein the oligonucleotide adapter in step h) comprises a barcode and optionally an UNIT

10. The method according to claim 8, wherein the size of the size selected PCR products is between 150 bp and 1000 bp, preferably the size of the selected PCR products is between 300-450.

11. The method according to claim 7, wherein an adapter is ligated to at least one of: the RNA provided in the sample of step a); the single-stranded cDNA obtained in step d); the double-stranded cDNA obtained in step e); the aRNA obtained in step f); and the cDNA obtained in step i).

12. A method for obtaining RNA sequence information from a cell, preferably a single cell, comprising the steps of 1) preparing a sequencing library as defined in claim 1; 2) optionally pooling one or more sequencing libraries prepared in step 1); and 3) sequencing, preferably deep-sequencing, the sequencing library.

13. A cDNA library comprising a barcode and a UMI obtainable by the methods of claim 1.

14. The cDNA library according to claim 13, wherein the cDNA library is further processed to be sequenced.

Description

DESCRIPTION OF THE FIGURES

[0118] FIG. 1: Vast transcriptome Analysis in Single cells by A-tailing (VASA-seq). Schematic representation of the method according to one embodiment of the invention. 1) Cell sorting, 2) Cell lysis, 3) RNA fragmentation, 4) A-tailing, 5) Reverse transcription (Barcode+UMI), 6) IVT and rRNA depletion, 8) Modified Cel-seq/Cel-seq2 protocol (sequencing libraries), 9) Sequencing, 10A) Alignment and gene counting, 10B) Isoform quantification and 10C) snRNA variant analysis.

[0119] FIG. 2: comparison of the VASA-seq methods to other single cell sequencing methods. A) schematic representation of the advantages of VASA-seq compared to the known methods RamDa-seq, Smart-seq2 and Cel-Seq. B) detected RNA species in mouse embryonic stem cells (mESC). C) Number of detected genes in mESC (130,000 read per cell) D) Gene body coverage E) Detected ncRNAs with VASAseq.

[0120] FIG. 3: A) 376 cultured mouse embryonic stem cells sorted into 384-plates containing mineral oil and barcoded primers (one barcode per well). Plates were stored at −80° C. before processing with VASA-seq. Final DNA libraries were sequenced to a depth of ˜85 million reads on the Illumina NextSeq instrument. Data was mapped with STAR and unique fragments and genes were obtained. Histogram shows the number of genes detected per cell. Low quality cells were filtered away, 316 out of the 376 were kept. B) Average coverage across the gene body of all protein coding genes. A flat line, as mostly seen for VASA-seq indicates even coverage across the whole transcript from 5′- to 3′-end. Even coverage is needed for full-length detection of transcripts. VASA-seq exhibit a slight bias at the 5′-end but overall shows even coverage. Data from CEL-Seq2 (K562 cells) are used as reference for a method with clear 3′-end bias. C) Barplots showing detection of different RNA species (rRNA excluded). Left plot shows that approximately 10% of the detected fragments in VASA-seq are derived from Non-coding RNAs. This does not correspond to unique molecules as protein coding genes are for example around 10× longer than small Non-coding RNAs. Right plot shows the fractions of different types of Non-coding RNA, out of the 10% shown in the left plot.

[0121] FIG. 4: A) 376 nuclei, dissociated from whole mouse brain, were sorted into 384-plates containing mineral oil and barcoded primers (one barcode per well). Plates were stored at −80° C. before processing with VASA-seq. Final DNA libraries were sequenced to a depth of ˜48 million reads on the Illumina NextSeq instrument. Data was mapped with STAR and unique fragments and genes were obtained. Histogram shows the number of genes detected per nuclei. Low quality nuclei were filtered away, 222 out of the 376 were kept. B) 376 nuclei, dissociated from the subventricular zone of a post mortem human brain sample, were sorted into 384-plates containing mineral oil and barcoded primers (one barcode per well). Plates were stored at −80° C. before processing with VASA-seq. Final DNA libraries were sequenced to a depth of ˜59 million reads on the Illumina NextSeq instrument. Data was mapped with STAR and unique fragments and genes were obtained. Histogram shows the number of genes detected per nuclei. Low quality nuclei were filtered away, 211 out of the 376 were kept. C) Mouse nuclei were separated in t-SNE space based on total gene expression. Each dot represents one nucleus. Several clusters/groups, representing different cells types, can be seen in the plot. Known markers for Neurons (top), Astrocytes (middle) and Oligodendrocytes (bottom) are visualized, red color indicates high expression of selected genes and blue color low expression. D) Human nuclei were separated in t-SNE space based on total gene expression. Each dot represents one nucleus. Several clusters/groups, representing different cells types, can be seen in the plot. Known markers for Neurons (top), Astrocytes (middle) and Oligodendrocytes (bottom) are visualized, red color indicates high expression of selected genes and blue color low expression.

EXAMPLES

Material and Methods

Part 1:

[0122] 1. Sorting and Lysis [0123] Sort cells in 384-plates with mineral oil and Cel-seq2 primers. [0124] Spin plates for 2 min at 4° C. (2000 rcf). [0125] Lyse cells at 65° C. for 5 minutes. Spin down and cool on ice.

[0126] 2. Fragmentation [0127] Dispense 50 nl of the following Fragmentation mix (add 26 μl per strip tube):

TABLE-US-00001 1x in nl 4 plates in μl ERCC Spike in 1:2.500 1 4.5 (dilute 1:50 from 1:50) dNTP 100 mM (25 mM each) 1 4.5 Invitrogen 5x FS buffer 48 221 [0128] Spin plate for 2 min at 4° C. (2000 rcf). [0129] Fragmentize RNA at 85° C. for 5 minutes. Spin down and cool on ice.

[0130] 3. End repair [0131] Dispense 150 nl of the following End repair mix (add 44 μl per strip tube):

TABLE-US-00002 Reagent 1x in nl 4 plates in μl H.sub.20 112.5 270 NEB T4 PNK 12.5 30 Invitrogen RNaseOUT 12.5 30 Invitrogen DTT (0.1M) 12.5 30 [0132] Spin plate for 2 min at 4° C. (2000 rcf) [0133] Incubate at 37° C. for 40 minutes. Spin down and cool on ice.

[0134] 4. Poly-A Tailing [0135] Dispense 50 nl of the following mix (add 26 μl per strip tube):

TABLE-US-00003 Reagent 1x in nl 4 plates in μl Invitrogen 5x FS buffer 23 106 Poly (A) Polymerase 1.5 7 ATP (10 mM-20% NH2 15 69 ATP) H.sub.20 10.5 48 [0136] Spin plate for 2 min at 4° C. (2000 rcf). [0137] Incubate at 37° C. for 15 min. Spin down and cool on ice

[0138] 5. cDNA Synthesis/Reverse Transcription (RT) [0139] Dispense 50 nl of the following mix to two plates (add 26 μl per strip tube):

TABLE-US-00004 Reagent 1x in nl 4 plates in μl Invitrogen DTT (0.1M) 5 23 Invitrogen SS III 17.5 81 H.sub.20 27.5 126 [0140] Spin plate for 2 min at 4° C. (2000 rcf). [0141] Incubate at 50° C. for 1 h. [0142] Spin down and cool on ice.

[0143] 6. Second Strand Synthesis [0144] Add 1920 nl (dispense 4×480 nl, place on ice after each dispension) of the following second strand mix (add 120 μl per strip tube, 4× strips):

TABLE-US-00005 Reagent 1x in nl 4 plates in μl H.sub.20 1347.5 2960 Invitrogen 2.sup.nd strand buffer 437.5 960 dNTP 10 mM 43.75 96 Invitrogen E.coli ligase 15.75 34.8 Invitrogen E.coli DNA 61.25 134.4 polymerase I Invitrogen RNAseH 15.75 34.8 [0145] Spin plate for 2 min at 4° C. (2000 rcf) [0146] Incubate at 16° C. for 2 hours. [0147] Move plates to ice. [0148] Set a thermocycler to 75° C. [0149] Incubate plates at 75° C. for 20 min. [0150] Make plastic boxes and spin down the plates (1 min at 300 rcf). [0151] Add 2×1000 μl mineral oil and collect all liquid “bubbles” in one 2 ml Eppendorf tube per sample. [0152] Spin down the tubes to separate the oil and aRNA. Then pipette all aRNA into a new 2 ml eppendorf tube, make sure not to get oil (the whole volume is 800 μl).

[0153] 7. Pool&Cleanup [0154] Warm AMPure XP beads to room temperature. [0155] Add 1× volume (approx. 800 μl) of mixed Ampure XP beads with bead binding buffer (diluted 1:8). [0156] Incubate 15 min at RT. [0157] Incubate on magnet stand for 5 min or until liquid is clear. Twist to get better “bands”. [0158] Remove supernatant without disturbing the beads. [0159] Wash pellet carefully with 1000 μl 80% ETOH. Incubate at least 30 seconds. [0160] Repeat above step. [0161] Remove as much ETOH as possible. [0162] Dry at RT for approx. 10 minutes or until dry. [0163] Elute RNA from beads with 6.4 μl water, start with one tube and use the material to elute from the other.

[0164] 8. IVT [0165] Add 9.6 μl of IVT mix per sample as in CEL-Seq2. Mix well and transfer everything to a 0.5 ml tube.

TABLE-US-00006 Reagent 1x reaction 4 + 1x reactions Ambion T7 buffer 1.6 μl 8 μl Ambion T7 enzyme 1.6 μl 8 μl Ambion ATP 1.6 μl 8 μl Ambion CTP 1.6 μl 8 μl Ambion GTP 1.6 μl 8 μl Ambion UTP 1.6 μl 8 μl [0166] Incubate at 37° C. for 14 hours, with lid at 70° C. Set cycler to go to 4° C. at end of incubation.

[0167] 9. DNA cleanup

[0168] EXO-SAP (to remove primers): [0169] Add 6 μl EXO-SAP to all tubes. [0170] Incubate at 15 minutes at 37° C., cool on ice.

[0171] 10. Pool&Cleanup [0172] Warm AMPure XP beads to room temperature. [0173] Add 40 μl of mixed Ampure XP beads to 1.5 ml tubes. [0174] Transfer samples (22 μl) to 1.5 ml eppendorf tubes. [0175] Incubate 15 min at RT. [0176] Incubate on magnet stand for 5 min or until liquid is clear. [0177] Remove supernatant without disturbing the beads. [0178] Wash pellet carefully with 1000 μl 80% ETOH. Incubate at least 30 seconds. [0179] Repeat above step. [0180] Remove as much ETOH as possible. [0181] Dry at RT for approx. 15 minutes or until dry. [0182] Elute RNA from beads with 24 μl water. [0183] Incubate away from magnet for at least 2 min. [0184] Incubate on magnet stand for 5 min or until liquid is clear. [0185] Transfer liquid to new tubes. [0186] Dilute samples 1:50 and run the Bioanalyzer RNA Pico total RNA. Also measure concentration with a Qubit. [0187] Then dilute samples to ˜100/μl.

Part 2:

[0188] 1. rRNA Depletion

[0189] Make Hyb-Mix (4+1 Reactions)

TABLE-US-00007 Reagent Volume Hyb-buffer 10 μl rRNA-dep-oligos (25 μM) 10 μl [0190] Add 4 μl mix to 6 μl of aRNA. [0191] Spin down and cool on ice. [0192] Incubate at 95° C. for 2 minutes, then decrease the temperature to 45° C. at a rate of 0.1° C./s.

[0193] Make RNase-Mix (4+1 Reactions)

TABLE-US-00008 Reagent Volume Epicentre RNaseH 10 μl (Thermostable) RNase buffer 40 μl [0194] Add 10 μl of RNase-mix to the Hyb-mix while keeping it on 45° C. [0195] Incubate at 45° C. for 30 minutes. [0196] Spin down and cool on ice.

[0197] Make DNase-Mix (4+1 Reactions)

TABLE-US-00009 Reagent Volume Promega DNase 10 μl Promega 10xBuffer 11 μl [0198] Add 4.2 μl of DNase-mix to the reaction. [0199] Incubate at 37° C. for 30 minutes. [0200] Spin down and cool on ice.

[0201] 2. Pool&Cleanup [0202] Warm AMPure XP beads to room temperature. [0203] Add 44 μl of Ampure XP beads to 1.5 ml tubes. [0204] Transfer samples (24.2 μl) to 1.5 ml eppendorf tubes. [0205] Incubate 15 min at RT. [0206] Incubate on magnet stand for 5 min or until liquid is clear. [0207] Remove supernatant without disturbing the beads. [0208] Wash pellet carefully with 1000 μl 80% ETOH. Incubate at least 30 seconds. [0209] Repeat above step. [0210] Remove as much ETOH as possible. [0211] Dry at RT for approx. 10 minutes or until dry. [0212] Elute RNA from beads with 6 μl water. [0213] Incubate away from magnet for at least 2 min. [0214] Incubate on magnet stand for 5 min or until liquid is clear. [0215] Transfer liquid to new tubes.

[0216] 3. Adapter Ligand [0217] Set a thermo cycler to 70° C. (with heated lid at 105° C.) and prepare the four 0.2 ml low binding tubes with the following, mix well by pipetting (keep cold):

TABLE-US-00010 a. rRNA depleted aRNA 5 μl a. 3′_adapter (20 μM) 1 μl [0218] Heat adapter-sample-mix at 70° C. for 2 min and then directly put on ice. [0219] Set a thermo cycler to 25° C. (with lid at 25° C. or without the lid closed) and prepare a 1.5 ml eppendorf tube with the following reagents, mix well by pipetting (keep cold):

TABLE-US-00011 i. NEB 10x T4 RNA Ligase Reaction Buffer 4.5 μl ii. NEB T4 RNA Ligase 2, truncated 4.5 μl iii. Invitrogen RNaseOUT 4.5 μl iv. H.sub.2O 4.5 μl [0220] Add 4 μl to each of the four 0.2 ml tubes, mix well by pipetting (keep cold). [0221] Spin down. Incubate at 25° C. for 1 h followed by 4° C. for (thermo cycler with lid at 25° C. or without the lid closed).

[0222] 4. 2.sup.nd cDNA Synthesis [0223] Set a thermo cycler to 65° C. (with heated lid at 105° C.) and prepare the four 0.2 ml low binding tubes with the following, mix well by pipetting (keep cold):

TABLE-US-00012 i. Adapter ligated RNA 10 μl  ii. dNTP Mix (10 mM each) 1 μl iii. RT primer (20 μM) 2 μl [0224] Heat at 65° C. for 5 min and then directly put on ice. [0225] Mix the following in a 1.5 ml Eppendorf tube, mix well by pipetting (keep cold):

TABLE-US-00013 v. Invitrogen 5x FS Buffer  18 μl vi. H.sub.2O 4.5 μl vii. Invitrogen 0.1M DTT 4.5 μl viii. Invitrogen RNaseOUT (40 U/μl) 4.5 μl ix. Invitrogen SS III (200 u/μl) 4.5 μl [0226] Add 8 μl to each of the four 0.2 ml tubes, mix well by pipetting (keep cold). [0227] Spin down. Incubate at 50° C. for 1 h followed by 70° C. for 15 min, 4° C. for (thermo cycler with heated lid at 85° C.).

[0228] 5. Strand Degradation [0229] Add 1 μl RNaseA (Thermo) to each tube. [0230] Incubate at 37° C. for 30 min, 4° C. for ∞ (thermo cycler with heated lid at 70° C.). [0231] Do bead cleanups as previously described but use 1:1 beads (22 μl) and elute in 20 μl water. [0232] Save first supernatant and mix with (22 μl) and elute in 20 μl water.

[0233] 6. PCR Amplification: [0234] Mix the following in a tube

TABLE-US-00014 H.sub.20 55 μl 2x NEBNext PCR mix 125 μl  RNA PCR Primer (RP1, from Illumina kit) 10 μl

[0235] Add to each tube:

TABLE-US-00015 Above mix 38 μl Index (one per sample)  2 μl Purified RT material 10 μl

[0236] Amplify the tube in the thermal cycler using the following PCR cycling conditions: [0237] 30 seconds at 98° C. [0238] 7-9 cycles of: [0239] 10 seconds at 98° C. [0240] 30 seconds at 60° C. [0241] 30 seconds at 72° C. [0242] 10 minutes at 72° C. [0243] Hold at 4° C.

[0244] 7. Size Selection [0245] Add 50 μl water to each sample. [0246] Add 50 μl bead to each sample, mix by pipetting (˜10 times). [0247] Bind for 5 min, then place on magnet. [0248] When clear, take supernatant and transfer to a new tube, then add 30 μl beads, mix by pipetting (˜10 times). The old beads you can throw away. [0249] Bind for 10 min, then place on magnet. [0250] When clear, remove supernatant wash beads with 1000 μl 80% EtOH. [0251] Repeat the wash one more time. [0252] Let beads dry, the elute in 25 μl water.

[0253] 8. Bead Cleanup [0254] Prewarm beads to room temperature. [0255] Vortex AMPure XP Beads until well dispersed, then add 20 μl to the 25 μl sample. Mix entire volume up ten times to mix thoroughly. [0256] Incubate at room temperature for 10 min. [0257] Place on magnetic stand for at least 5 min, until liquid appears clear. [0258] Remove and discard the supernatant. [0259] Add 1000 μl freshly prepared 80% EtOH. [0260] Incubate at least 30 seconds, then remove and discard supernatant without disturbing beads. [0261] Add 1000 μl freshly prepared 80% EtOH [0262] Incubate at least 30 seconds, then remove and discard supernatant without disturbing beads. [0263] Air dry beads for 10 min, or until completely dry. [0264] Resuspend with 10 μl water. Pipette entire volume up and down ten times to mix thoroughly. [0265] Incubate at room temperature for 2 min. [0266] Place on magnetic stand for 5 min, until liquid appears clear. [0267] Run the Bioanalyzer DNA HS. Also measure concentration with a Qubit.

Example 1

[0268] For protein coding genes, VASA-seq detected about 10% more genes at the same sequencing depth compared to RamDa-seq (FIG. 2C). Compared to Cel-seq2 also and at the same sequencing depth, VASA-seq detected around 2.5× times more genes. For ncRNA (not including IncRNA), VASA-seq detects at least 10-20× more genes than RamDa-seq and Cel-seq2 (FIG. 2B). IncRNA are about the same for RamDa seq and V ASA-seq.